Insulin Derivative

ABSTRACT

The present invention relates to novel human insulin derivatives which are soluble at physiological pH values and have a prolonged profile of action. The invention also relates to methods of providing such derivatives, to pharmaceutical compositions containing them, to methods of treating diabetes and hyperglycaemia using the insulin derivatives of the invention and to the use of such insulin derivatives in the treatment of diabetes and hyperglycaemia.

FIELD OF THE INVENTION

The present invention relates to novel human insulin derivatives whichare soluble at physiological pH values and have a prolonged profile ofaction. The invention also relates to methods of providing suchderivatives, to pharmaceutical compositions containing them, to methodsof treating diabetes and hyperglycaemia using the insulin derivatives ofthe invention and to the use of such insulin derivatives in thetreatment of diabetes and hyperglycaemia.

BACKGROUND OF THE INVENTION

Currently, the treatment of diabetes, both type 1 diabetes and type 2diabetes, relies to an increasing extent on the so-called intensiveinsulin treatment. According to this regimen, the patients are treatedwith multiple daily insulin injections comprising one or two dailyinjections of a long acting insulin to cover the basal insulinrequirement supplemented by bolus injections of a rapid acting insulinto cover the insulin requirement related to meals.

Long acting insulin compositions are well known in the art. Thus, onemain type of long acting insulin compositions comprises injectableaqueous suspensions of insulin crystals or amorphous insulin. In thesecompositions, the insulin compounds utilized typically are protamineinsulin, zinc insulin or protamine zinc insulin.

Certain drawbacks are associated with the use of insulin suspensions.Thus, in order to secure an accurate dosing, the insulin particles mustbe suspended homogeneously by gentle shaking before a defined volume ofthe suspension is withdrawn from a vial or expelled from a cartridge.Also, for the storage of insulin suspensions, the temperature must bekept within more narrow limits than for insulin solutions in order toavoid lump formation or coagulation.

While it was earlier believed that protamines were non-immunogenic, ithas now turned out that protamines can be immunogenic in man and thattheir use for medical purposes may lead to formation of antibodies.Also, evidence has been found that the protamine-insulin complex isitself immunogenic. Therefore, with some patients the use of long actinginsulin compositions containing protamines must be avoided.

Another type of long acting insulin compositions are solutions having apH value below physiological pH from which the insulin will precipitatebecause of the rise in the pH value when the solution is injected. Adrawback with these solutions is that the particle size distribution ofthe precipitate formed in the tissue on injection, and thus the releaseprofile of the medication, depends on the blood flow at the injectionsite and other parameters in a somewhat unpredictable manner. A furtherdrawback is that the solid particles of the insulin may act as a localirritant causing inflammation of the tissue at the site of injection.

International patent application published under number WO 2005/012347(Novo Nordisk A/S) concerns insulin derivatives which have a sidechainattached to either the α-amino group of the N-terminal amino acidresidue of the B-chain or the ε-amino group of a Lys residue present inthe B chain.

International patent application No. EP2006/050593 (Novo Nordisk A/S)discloses insulin derivatives having an aromatic group in the sidechain.

Patent application no. EP2006/050594 (Novo Nordisk A/S), discloseinsulin derivatives having a PEG in the side chain.

Other insulin derivatives are disclosed in JP laid-open patentapplication No. 1-254699 (Kodama Co., Ltd.) and WO 95/07931 (NovoNordisk A/S).

However, there is still a need for insulin having a more prolongedprofile of action than the insulin derivatives known up till now.

SUMMARY OF THE INVENTION

In one aspect of the invention an insulin derivative is having a formula

wherein Ins is a parent insulin moiety andQ₁-Q₂-[CH₂]_(n)—X₁—[CH₂]_(n)-Q₃-[CH₂]_(n)—X₂—[CH₂]_(n)-Q₄-[CH₂]_(n)—X₃—[CH₂]_(n)-Q₅-[CH₂]_(n)-Zis a substituent and where the Ins is attached to the substituent via anamide bond between the α-amino group of the N-terminal amino acidresidue of the B chain of Ins or an ε-amino group of a Lys residuepresent in the A or B chain of Ins and a CO group in Q₁ or Q₂ of thesubstituent;each n is independently 0, 1, 2, 3, 4, 5 or 6;

Q₁ is:

-   -   an amino acid amide of an amino acid with a carboxylic acid in        the side chain, or an amino acid with an uncharged side chain,        which residue forms, with its carboxylic acid group, an amide        group together with the α-amino group of the N-terminal amino        acid residue of the B chain of Ins or together with the ε-amino        group of a Lys residue present in the A or B chain of Ins, or

a chain composed of two, three or four α-amino acid amide or amino acidresidues as specified above linked together via amide bonds, whichchain—via an amide bond—is linked to the α-amino group of the N-terminalamino acid residue of the B chain of Ins or to the ε-amino group of aLys residue present in the A or B chain of Ins, or

a bond

Q₂ is:

—COCH(CONH₂)—

—COCH₂N(CH₂CONH₂)—

—COCH₂N(CH₂CONH₂)COCH₂N(CH₂CONH₂)

—COCH₂CH₂N(CH₂CH₂CONH₂)—

—COCH₂CH₂N(CH₂CH₂CONH₂)—COCH₂CH₂N(CH₂CH₂CONH₂)—

—COCH₂N(CH₂CH₂CONH₂)—

—COCH₂CH₂N(CH₂CONH₂)—

—COCH₂OCH₂CONH—

—CO—((CR⁵R⁶)₁₋₆—NH—CO)₁₋₄—;

—CO—((CR⁵R⁶)₁₋₆—CO—NH)₁₋₄—, where R⁵ independently can be H, —CH₃,—(CH₂)₁₋₆CH₃ or —CONH₂ and R⁶ independently can be H, —CH₃,—(CH₂)₁₋₆CH₃; or

a bond

-   -   provided that        -   at least one of Q₁ or Q₂ is not a bond, and        -   that Q₂ is not —CO—(CH₂)₂—CO—NH— when n is 0 or 1, X₁ is a            bond and Q₃ is (CH₂CH₂O)₂—, (CH₂CH₂O)₃— or            (CH₂CH₂OCH₂CH₂CH₂CH₂O)— and        -   that if an amine in Q₁ or Q₂ forms a bond with the rest of            the substituent, the amine must be bound to the rest of the            substituent via a carbonyl group;            Q₃, Q₄, and Q₅ independently of each other can be

—(CH₂)_(m)— where m is an integer in the range of 6 to 32;

a divalent hydrocarbon chain comprising 1, 2 or 3 —CH═CH— groups and anumber of —CH₂— groups sufficient to give a total number of carbon atomsin the chain in the range of 4 to 32;

—CO—((CR⁵R⁶)₁₋₆—NH—CO)—;

—(CO—(CR⁵R⁶)₁₋₆—CO—NH)₁₋₄—, where R⁵ independently can be H, —CH₃,—(CH₂)₁₋₆CH₃ or —CONH₂ and R⁶ independently can be H, —CH₃,—(CH₂)₁₋₆CH₃;

—CO—(CH₂)₀₋₃—Ar—(CH₂)₀₋₃— where Ar can be arylene or heteroarylene,which may be substituted with one or two groups selected from the groupconsisting of —CH₃, —(CH)₁₋₆—CH₃, —CONR¹R² or —SO₂NR¹R² where R¹ and R²independently of each other can be H, —CH₃ or —(CH)₁₋₆—CH₃;

(CH₂CH₂O)_(y)—; (CH₂CH₂CH₂O)_(y)—; (CH₂CH₂CH₂CH₂O)_(y)—;(CH₂CH₂OCH₂CH₂CH₂CH₂O)_(y)— or (CH₂CH₂CH₂OCH₂CH₂CH₂CH₂O)_(y)—;—(CH₂OCH₂)_(y)— where y is 1-20;

arylene or heteroarylene, which may be substituted with one or twogroups selected from the group consisting of —CH₃, —(CH)₁₋₆—CH₃,—CONR¹R² or —SO₂NR¹R² where R¹ and R², independently of each other canbe H, —CH₃ or —(CH)₁₋₆—CH₃;

a chain of the formula

—(CH₂)_(s)—Y₁—(Ar)_(v1)—Y₂—(CH₂)_(w)—Y₃—(Ar)_(v2)—Y₄—(CH₂)_(t)—Y₅—(Ar)_(v3)—Y₆—(CH₂)_(z)—

wherein Ar is defined as above, Y₁-Y₆ independently of each other can beO, S, S═O, SO₂ or a bond; where s, w, t and z independently of eachother are zero or an integer from 1 to 10 so that the sum of s, w, t andz is in the range from 4 to 30, and v₁, v₂, and v₃ independently of eachother can be zero or 1 with the proviso that Y₁-Y₆ do not link to eachother and that the structure —O—(CH₂)₁—O— does not occur; or

a bond;

with the proviso that at least one of Q₃-Q₅ is not a bond;X₁, X₂ and X₃ are independently of each other

O;

—C═O

a bond;

NCOR¹, where R¹ can be H, —CH₃ or —(CH)₁₋₆—CH₃; or

where R is hydrogen, C₁₋₃-alkyl, C₂₋₃-alkenyl or C₂₋₃-alkynyl;

with the proviso that X₁, X₂ and X₃ cannot bind to Z and when X₁, X₂ andX₃ are O, then X₁, X₂ and X₃ do not bind directly to O in Q₃, Q₄, and Q₅

and

Z is:

—COOH;

—CO-Asp;

—CO-Glu;

—CO-Gly;

—CO-Sar;

—CH(COOH)₂;

—N(CH₂COOH)₂;

—SO₃H

—OSO₃H

—OPO3H₂

—PO₃H₂ or

-tetrazol-5-yl or

—O—W₁,

where W₁ is arylene or heteroarylene, which may be substituted with oneor two groups selected from the group consisting of tetrazo-5-lyl,—COOH, —SO₃H, —(CH₂)₁₋₆—SO₃H, —(CH₂)₁₋₆—O—PO₃H₂, —CONR³R⁴ or —SO₂NR³R⁴where R³ and R⁴, independently of each other can be H, —(CH₂)₁₋₆—SO₃H,or —(CH₂)₁₋₆—O—PO₃H₂; provided that when Z is —O—W₁ then Q₁ must bepresentand any Zn²⁺ complex thereof

In one aspect of the invention Q₂ of the insulin derivative is selectedfrom the group consisting of

—COCH(CONH₂)—

—COCH₂N(CH₂CONH₂)—

—COCH₂N(CH₂CONH₂)COCH₂N(CH₂CONH₂)

—COCH₂CH₂N(CH₂CH₂CONH₂)—

—COCH₂CH₂N(CH₂CH₂CONH₂)—COCH₂CH₂N(CH₂CH₂CONH₂)—

—COCH₂N(CH₂CH₂CONH₂)—

—COCH₂CH₂N(CH₂CONH₂)—

—COCH₂OCH₂CONH—;

—CO—((CR⁵R⁶)₁₋₆—NH—CO)₁₋₄—; or

—CO—((CR⁵R⁶)₁₋₆—CO—NH)₁₋₄—, where R⁵ independently can be H, —CH₃,—(CH₂)₁₋₆CH₃ or —CONH₂ and R⁶ independently can be H, —CH₃, —(CH₂)₁₋₆CH₃

In one aspect of the invention Q₃ is —(CH₂)_(m)— where m is an integerin the range of 6 to 32 or from 8 to 20 or m is 12, 13, 14, 15 or 16.

In one aspect of the invention Q₁, Q₄, Q₅, X₁, X₂ and X₃ is bonds and nis zero.

In one aspect of the invention Q₃ is —(CH₂)_(m)— where m is an integerin the range of 6 to 32 or from 8 to 20 or m is 12, 13, 14, 15 or 16,Q₁, Q₄, Q₅, X₁, X₂ and X₃ is bonds, n is zero and Z is —COOH.

In one aspect of the invention one of Q₃, Q₄, or Q₅ is (CH₂CH₂O)_(y)—;(CH₂CH₂CH₂O)_(y)—; (CH₂CH₂CH₂CH₂O)_(y)—; (CH₂CH₂OCH₂CH₂CH₂CH₂O)_(y)— or(CH₂CH₂CH₂OCH₂CH₂CH₂CH₂O)_(y)—; —(CH₂OCH₂)_(y)— where y is 1-20.

In one aspect of the invention Q₃ is

—CO—((CR⁵R⁶)₁₋₆—NH—CO)—;

—(CO—(CR⁵R⁶)₁₋₆—CO—NH)₁₋₄—, where R⁵ independently can be H, —CH₃,—(CH₂)₁₋₆CH₃ or —CONH₂ and R⁶ independently can be H, —CH₃,—(CH₂)₁₋₆CH₃;

—CO—(CH₂)₀₋₃—Ar—(CH₂)₀₋₃— where Ar can be arylene or heteroarylene,which may be substituted with one or two groups selected from the groupconsisting of —CH₃, —(CH)₁₋₆—CH₃, —CONR¹R² or —SO₂NR¹R² where R¹ and R²independently of each other can be H, —CH₃ or —(CH)₁₋₆—CH₃; or

a bond

Q₄ is

—(CH₂)_(m)— where m is an integer from 4 to 22;

a divalent hydrocarbon chain comprising 1, 2 or 3 —CH═CH— groups and anumber of —CH₂— groups sufficient to give a total number of carbon atomsin the chain in the range of 4 to 22;

arylene or heteroarylene, which may be substituted with one or twogroups selected from the group consisting of —CH₃, —(CH)₁₋₆—CH₃,—CONR¹R² or —SO₂NR¹R² where R¹ and R², independently of each other canbe H, —CH₃ or —(CH)₁₋₆—CH₃; or

-   -   a chain of the formula

—(CH₂)_(s)—Y₁—(Ar)_(v1)—Y₂—(CH₂)_(w)—Y₃—(Ar)_(v2)—Y₄—(CH₂)_(t)—Y₅—(Ar)_(v3)—Y₆—(CH₂)_(z)—

wherein Ar is defined as above, Y₁-Y₆ independently of each other can beO, S, S═O, SO₂ or a bond; where s, w, t and z independently of eachother are zero or an integer from 1 to 10 so that the sum of s, w, t andz is in the range from 4 to 30, and v₁, v₂, and v₃ independently of eachother can be zero or 1 with the proviso that Y₁-Y₆ do not link to eachother and that the structure —O—(CH₂)₁—O— does not occur;

X₁ is

O;

—C═O

NCOR¹, where R¹ can be H, —CH₃ or —(CH)₁₋₆—CH₃; or

-   -   where R is hydrogen, C₁₋₃-alkyl, C₂₋₃-alkenyl or C₂₋₃-alkynyl;    -   with the proviso that when X₁ is O then X₁ does not bind        directly to O in Q₄;        X₂, X₃ and Q₅ are bonds;        All values of n are zero; and

Z is:

—COOH;

—CO-Asp;

—CO-Glu;

—CO-Gly;

—CO-Sar;

—CH(COOH)₂;

—N(CH₂COOH)₂;

—SO₃H

—OSO₃H

—OPO3H₂

—PO₃H₂ or

-tetrazol-5-yl or

—O—W₁,

where W₁ is arylene or heteroarylene, which may be substituted with oneor two groups selected from the group consisting of tetrazo-5-lyl,—COOH, —SO₃H, —(CH₂)₁₋₆—SO₃H, —(CH₂)₁₋₆—O—PO₃H₂, —CONR³R⁴ or —SO₂NR³R⁴where R³ and R⁴ independently of each other can be H, —(CH₂)₁₋₆—SO₃H, or—(CH₂)₁₋₆—O—PO₃H₂; and any Zn²⁺ complex thereof.

In one aspect of the invention Z is —COOH.

In one aspect of the invention the parent insulin of the insulinderivative is an insulin analogue.

In one aspect of the invention the parent insulin is selected from thegroup consisting of: desB30 human insulin, GlyA21 human insulin,GlyA21desB30 human insulin, GlyA21ArgB31ArgB32 human insulin,LysB3GluB29 human insulin, LysB28ProB29 human insulin and ThrB29LysB30human insulin.

In one aspect of the invention there is provided a pharmaceuticalcomposition for the treatment of diabetes in a patient in need of suchtreatment, comprising a therapeutically effective amount of an insulinderivative according to the invention.

In one aspect of the invention there is provided a method for producinga pharmaceutical composition comprising a therapeutically effectiveamount of an insulin derivative according to the invention, wherein upto about 10 zinc atoms per 6 molecules of insulin derivative are addedto the pharmaceutical composition.

In one aspect of the invention there is provided a method of treatingdiabetes in a patient in need of such a treatment, comprisingadministering to the patient a therapeutically effective amount of aninsulin derivative according to the invention.

In one aspect of the invention the insulin derivative is administeredpulmonary.

In one aspect of the invention there is provided a mixture of an insulinderivative according to the invention and a rapid acting insulinanalogue selected from the group consisting of AspB28 human insulin;LysB28ProB29 human insulin and LysB3GluB29 human insulin.

In one aspect of the invention the insulin derivative is selected fromthe group consisting of:

-   N^(εB29)-ω-carboxy-pentadecanoyl-γ-L-glutamylamide desB30 human    insulin,-   N^(εB29)-ω-carboxy-pentadecanoyl-γ-amino-butanoyl desB30 human    insulin,-   N^(εB29)-ω-carboxy-tetradecanoyl-γ-L-glutamylamide desB30 human    insulin,-   N^(εB29)-ω-carboxy-tridecanoyl-γ-L-glutamylamide desB30 human    insulin,-   N^(εB29)-ω-carboxy-pentadecanoyl-β-alanyl desB30 human insulin,-   N^(εB29)-ω-carboxy-pentadecanoyl-γ-L-aspartylamide desB30 human    insulin,-   N^(εB29)-ω-carboxy-pentadecanoyl-ε-aminohexanoyl desB30 human    insulin,-   N^(εB29)-ω-carboxy-pentadecanoyl-δ-aminopentanoyl desB30 human    insulin,-   N^(εB29)-10-(4-carboxyphenoxy)-decanoyl-γ-L-glutamylamide desB30    human insulin,-   N^(εB29)-4-[11-(4-Carboxyphenyl)undecanoylamino]butyryl desB30 human    insulin,-   N^(εB29)-(3-(3-{4-[3-(7-carboxyheptanoylamino)propoxy]butoxy}propylcarbamoyl)-propionyl-γ-glutamylamide)    desB30 human Insulin,-   N^(εB29)-ω-carboxy-tridecanoyl-γ-amino-butanoyl desB30 human    insulin,-   N^(εB29)-ω-carboxy-undecanoyl-γ-amino-butanoyl desB30 human insulin,-   N^(εB29)-ω-carboxy-tetradecanoyl-γ-amino-butanoyl desB30 human    insulin,-   N^(εB29)-{4-[10-(4-Carboxy-phenoxy)-decanoylamino]-butyryl}desB30    insulin,-   N^(εB29)-{4-[(14-Carboxy-tetradecanoylamino)-methyl]-benzoyl}desB30    insulin,-   N^(εB29)-[16-(4-Carboxy-phenoxy)-hexadecanoyl]desB30 insulin,-   N^(εB29)-{4-[(15-carboxypentadecanoylamino)benzoyl]-desB30 human    insulin and-   N^(εB29)-{4-[(15-Carboxy-pentadecanoylamino)-methyl]-benzoyl}-desB30    insulin

DEFINITIONS

By “insulin analogue” as used herein is meant a polypeptide which has amolecular structure which formally can be derived from the structure ofa naturally occurring insulin, for example that of human insulin, bydeleting and/or exchanging at least one amino acid residue occurring inthe naturally occurring insulin and/or adding at least one amino acidresidue. The added and/or exchanged amino acid residues can either becodable amino acid residues or other naturally occurring residues orpurely synthetic amino acid residues.

In aspects of the invention a maximum of 17 amino acids have beenmodified. In aspects of the invention a maximum of 15 amino acids havebeen modified. In aspects of the invention a maximum of 10 amino acidshave been modified. In aspects of the invention a maximum of 8 aminoacids have been modified. In aspects of the invention a maximum of 7amino acids have been modified. In aspects of the invention a maximum of6 amino acids have been modified. In aspects of the invention a maximumof 5 amino acids have been modified. In aspects of the invention amaximum of 4 amino acids have been modified. In aspects of the inventiona maximum of 3 amino acids have been modified. In aspects of theinvention a maximum of 2 amino acids have been modified. In aspects ofthe invention 1 amino acid has been modified.

With “desB30 insulin”, “desB30 human insulin” is meant a natural insulinor an analogue thereof lacking the B30 amino acid residue. Similarly,“desB29desB30 insulin” or “desB29desB30 human insulin” means a naturalinsulin or an analogue thereof lacking the B29 and B30 amino acidresidues.

With “B1”, “A1” etc. is meant the amino acid residue at position 1 inthe B-chain of insulin (counted from the N-terminal end) and the aminoacid residue at position 1 in the A-chain of insulin (counted from theN-terminal end), respectively. The amino acid residue in a specificposition may also be denoted as e.g. PheB1 which means that the aminoacid residue at position B1 is a phenylalanine residue.

With “insulin” as used herein is meant human insulin, porcine insulin orbovine insulin with disulfide bridges between CysA7 and CysB7 andbetween CysA20 and CysB19 and an internal disulfide bridge between CysA6and CysA11.

By “parent insulin” is meant a naturally occurring insulin such as humaninsulin or porcine insulin. Alternatively, the parent insulin can be aninsulin analogue.

The expression “uncharged” means that no group or groups that wouldassume a charge at pH interval 4 to 9 are present. For example no freecarboxylic acids are present.

With “fatty difunctionalized moiety” is meant a carbon chain of 6 to 32carbon atoms comprising two functional groups selected from carboxy,amino or hydroxyl.

The term “non-linking amide” is meant to describe an amide functionpresent in a side chain or side group of a residue present in thesubstituent, such that said amide bond is not used to connect theresidues of the substituent together. It should be understood that aresidue of the substituent in addition to the non-linking amide cancomprise further amide groups, eg. amides that bind to other residues ofthe substituent.

“Amino acid amide residue” means the alpha-carboxy amide of an aminoacid, or if the amino acid contains a carboxylic acid in the side-chain,“amino acid amide” means amide of either the alpha-carboxy group, oramide of the side-chain carboxy group, as specified.

When an insulin derivative according to the invention is stated to be“soluble at physiological pH values” it means that the insulinderivative can be used for preparing insulin compositions that are fullydissolved at physiological pH values. Such favourable solubility mayeither be due to the inherent properties of the insulin derivative aloneor a result of a favourable interaction between the insulin derivativeand one or more ingredients contained in the vehicle.

The term “no blunting” as used herein means that when formulated in oneformulation both the rapid acting insulin and the acylated insulin hasprofile of action which is identical or substantially identical with theprofile of action, when administering the rapid acting insulin and theacylated insulin in separate formulations.

The expression “high molecular weight insulin” or “hmw” means that themolecular weight of a complex of human insulin, of an insulin analogueor of an insulin derivative is above human serum albumin, above adodecameric complex of an insulin analogue or of an insulin derivativeor more than about 72 kDalton.

The expression “medium molecular weight insulin” or “mmw” means that themolecular weight of a complex of human insulin, of an insulin analogueor of an insulin derivative is from about an insulin hexamer to about aninsulin dodecamer between 24 and 80 kDalton.

The expression “low molecular weight insulin” or “lmw” means that themolecular weight of a human insulin, an insulin analogue or an insulinderivative is below 24 kDalton.

The following abbreviations have been used in the specification andexamples:

Cv column volume

HPLC High Performance Liquid Chromatography

HSA human serum albumin

LC liquid chromatography

MALDI Matrix Assisted Laser Desorption Ionization

MS mass spectrometry

RT room temperature

SEC size exclusion chromatography

SPA Scitillation Proximity Assay

Tris tris(hydroxymethyl)aminomethane

O.D. optical density=absorbance

X2 monomer AspB9 GluB27 human insulin

DIEA: N,N-diisopropylethylamine

DMF: N,N-dimethylformamide

Sar: Sarcosine (N-methyl-glycine)

tBu: tert-butyl

TSTU: O—(N-succinimidyl)-1,1,3,3-tetramethyluronium tetrafluoroborate

THF: Tetrahydrofuran

EtOAc: Ethyl acetate

DIPEA: Diisopropylethylamine TEA: triethyl amine

TFA: trifluoracetic acid

DCM: dichloromethane

RT: room temperature

PEG: polyethyleneglycol

GIR: Glucose infusion rate

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference in theirentirety and to the same extent as if each reference were individuallyand specifically indicated to be incorporated by reference and were setforth in its entirety herein (to the maximum extent permitted by law).

All headings and sub-headings are used herein for convenience only andshould not be construed as limiting the invention in any way.

The use of any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

The citation and incorporation of patent documents herein is done forconvenience only and does not reflect any view of the validity,patentability, and/or enforceability of such patent documents.

This invention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw.

DESCRIPTION OF THE INVENTION

The present invention is based on the recognition that having asubstituent in an insulin derivative molecule where only the terminalgroup is charged, plays an important role for the in vivo duration ofaction of prolonged-acting insulins, and for the mixability ofprolonged-acting insulin with fast-acting insulin with no blunting.

Advantageously, insulin derivatives according to the invention aresoluble at physiological pH values, have a potency which is comparableto that of human insulin, and are mixable with fast-acting insulins withno blunting. The individual profiles of action of mixed basal and bolusinsulins are retained in formulations containing Zn(II) concentrationsof up to or less than approximately 3 Zn(II) per insulin hexamer whichlimits the risk of precipitations in the formulation, compared toformulations containing more than 3 Zn (II) per insulin hexamer.

The invention is summarized in the following paragraphs:

An insulin derivative having a formula

wherein Ins is a parent insulin moiety andQ₁-Q₂-[CH₂]_(n)—X₁—[CH₂]_(n)-Q₃-[CH₂]_(n)—X₂—[CH₂]_(n)-Q₄-[CH₂]_(n)—X₃—[CH₂]_(n)-Q₅-[CH₂]_(n)-Zis a substituent and where the Ins is attached to the substituent via anamide bond between the α-amino group of the N-terminal amino acidresidue of the B chain of Ins or an ε-amino group of a Lys residuepresent in the A or B chain of Ins and a CO group in Q₁ or Q₂ of thesubstituent;each n is independently 0, 1, 2, 3, 4, 5 or 6;

Q₁ is:

-   -   an amino acid amide of an amino acid with a carboxylic acid in        the side chain, or an amino acid with an uncharged side chain,        which residue forms, with its carboxylic acid group, an amide        group together with the α-amino group of the N-terminal amino        acid residue of the B chain of Ins or together with the ε-amino        group of a Lys residue present in the A or B chain of Ins, or

a chain composed of two, three or four α-amino acid amide or amino acidresidues as specified above linked together via amide bonds, whichchain—via an amide bond—is linked to the α-amino group of the N-terminalamino acid residue of the B chain of Ins or to the ε-amino group of aLys residue present in the A or B chain of Ins, or

a bond

Q₂ is:

—COCH(CONH₂)—

—COCH₂N(CH₂CONH₂)—

—COCH₂N(CH₂CONH₂)COCH₂N(CH₂CONH₂)

—COCH₂CH₂N(CH₂CH₂CONH₂)—

—COCH₂CH₂N(CH₂CH₂CONH₂)—COCH₂CH₂N(CH₂CH₂CONH₂)—

—COCH₂N(CH₂CH₂CONH₂)—

—COCH₂CH₂N(CH₂CONH₂)—

—COCH₂OCH₂CONH—

—CO—((CR⁵R⁶)₁₋₆—NH—CO)₁₋₄—;

—CO—((CR⁵R⁶)₁₋₆—CO—NH)₁₋₄—, where R⁵ independently can be H, —CH₃,—(CH₂)₁₋₆CH₃ or —CONH₂ and R⁶ independently can be H, —CH₃,—(CH₂)₁₋₆CH₃; or

a bond

-   -   provided that        -   at least one of Q₁ or Q₂ is not a bond, and        -   that Q₂ is not —CO—(CH₂)₂—CO—NH— when n is 0 or 1, X₁ is a            bond and Q₃ is (CH₂CH₂O)₂—, (CH₂CH₂O)₃— or            (CH₂CH₂OCH₂CH₂CH₂CH₂O)— and        -   that if an amine in Q₁ or Q₂ forms a bond with the rest of            the substituent, the amine must be bound to the rest of the            substituent via a carbonyl group;            Q₃, Q₄, and Q₅ independently of each other can be

—(CH₂)_(m)— where m is an integer in the range of 6 to 32;

a divalent hydrocarbon chain comprising 1, 2 or 3 —CH═CH— groups and anumber of —CH₂— groups sufficient to give a total number of carbon atomsin the chain in the range of 4 to 32;

—CO—((CR⁵R⁶)₁₋₆—NH—CO)—;

—(CO—(CR⁵R⁶)₁₋₆—CO—NH)₁₋₄—, where R⁵ independently can be H, —CH₃,—(CH₂)₁₋₆CH₃ or —CONH₂ and R⁶ independently can be H, —CH₃,—(CH₂)₁₋₆CH₃;

—CO—(CH₂)₀₋₃—Ar—(CH₂)₀₋₃— where Ar can be arylene or heteroarylene,which may be substituted with one or two groups selected from the groupconsisting of —CH₃, —(CH)₁₋₆—CH₃, —CONR¹R² or —SO₂NR¹R² where R¹ and R²,independently of each other can be H, —CH₃ or —(CH)₁₋₆—CH₃;

(CH₂CH₂O)_(y)—; (CH₂CH₂CH₂O)_(y)—; (CH₂CH₂CH₂CH₂O)_(y)—;(CH₂CH₂OCH₂CH₂CH₂CH₂O)_(y)— or (CH₂CH₂CH₂OCH₂CH₂CH₂CH₂O)_(y)—;—(CH₂OCH₂)_(y)— where y is 1-20;

arylene or heteroarylene, which may be substituted with one or twogroups selected from the group consisting of —CH₃, —(CH)₁₋₆—CH₃,—CONR¹R² or —SO₂NR¹R² where R¹ and R², independently of each other canbe H, —CH₃ or —(CH)₁₋₆—CH₃; or

a chain of the formula

—(CH₂)_(s)—Y₁—(Ar)_(v1)—Y₂—(CH₂)_(w)—Y₃—(Ar)_(v2)—Y₄—(CH₂)_(t)—Y₅—(Ar)_(v3)—Y₆—(CH₂)_(z)—

wherein Ar is defined as above, Y₁-Y₆ independently of each other can beO, S, S═O, SO₂ or a bond; where s, w, t and z independently of eachother are zero or an integer from 1 to 10 so that the sum of s, w, t andz is in the range from 4 to 30, and v₁, v₂, and v₃ independently of eachother can be zero or 1 with the proviso that Y₁-Y₆ do not link to eachother and that the structure —O—(CH₂)₁—O— does not occur; or

a bond;

with the proviso that at least one of Q₃-Q₅ is not a bond;X₁, X₂ and X₃ are independently of each other

O;

—C═O

-   -   a bond;    -   NCOR¹, where R¹ can be H, —CH₃ or —(CH)₁₋₆—CH₃; or

where R is hydrogen, C₁₋₃-alkyl, C₂₋₃-alkenyl or C₂₋₃-alkynyl;

with the proviso that

-   -   —X₁, X₂ and X₃ cannot bind to Z and    -   when X₁, X₂ and X₃ are O then X₁, X₂ and X₃ do not bind directly        to O in Q₃, Q₄, and Q₅        and

Z is:

—COOH;

—CO-Asp;

—CO-Glu;

—CO-Gly;

—CO-Sar;

—CH(COOH)₂;

—N(CH₂COOH)₂;

—SO₃H

—OSO₃H

—OPO3H₂

—PO₃H₂ or

-tetrazol-5-yl or

—O—W₁,

where W₁ is arylene or heteroarylene, which may be substituted with oneor two groups selected from the group consisting of tetrazo-5-lyl,—COOH, —SO₃H, —(CH₂)₁₋₆—SO₃H, —(CH₂)₁₋₆—O—PO₃H₂, —CONR³R⁴ or —SO₂NR³R⁴where R³ and R⁴ independently of each other can be H, —(CH₂)₁₋₆—SO₃H, or—(CH₂)₁₋₆—O—PO₃H₂; provided that when Z is —O—W₁ then Q₁ must bepresent;and any Zn²⁺ complex thereof.

2. An insulin derivative having a formula

wherein Ins is a parent insulin moiety andQ₁-Q₂-[CH₂]_(n)—X₁—[CH₂]_(n)-Q₃-[CH₂]_(n)—X₂—[CH₂]_(n)-Q₄-[CH₂]_(n)—X₃—[CH₂]_(n)-Q₅-[CH₂]_(n)-Zis a substituent and where the Ins is attached to the substituent via anamide bond between the α-amino group of the N-terminal amino acidresidue of the B chain of Ins or an ε-amino group of a Lys residuepresent in the A or B chain of Ins and a CO group in Q₁ or Q₂ of thesubstituent;each n is independently 0, 1, 2, 3, 4, 5 or 6;

Q₁ is:

-   -   an amino acid amide of an amino acid with a carboxylic acid in        the side chain, or an amino acid with an uncharged side chain,        which residue forms, with its carboxylic acid group, an amide        group together with the α-amino group of the N-terminal amino        acid residue of the B chain of Ins or together with the ε-amino        group of a Lys residue present in the A or B chain of Ins, or

a chain composed of two, three or four α-amino acid amide or amino acidresidues as specified above linked together via amide bonds, whichchain—via an amide bond—is linked to the α-amino group of the N-terminalamino acid residue of the B chain of Ins or to the ε-amino group of aLys residue present in the A or B chain of Ins, or

a bond

Q₂ is:

—COCH(CONH₂)—

—COCH₂N(CH₂CONH₂)—

—COCH₂N(CH₂CONH₂)COCH₂N(CH₂CONH₂)

—COCH₂CH₂N(CH₂CH₂CONH₂)—

—COCH₂CH₂N(CH₂CH₂CONH₂)—COCH₂CH₂N(CH₂CH₂CONH₂)—

—COCH₂N(CH₂CH₂CONH₂)—

—COCH₂CH₂N(CH₂CONH₂)—

—COCH₂OCH₂CONH—

—CO—((CR⁵R⁶)₁₋₆—NH—CO)₁₋₄—;

—CO—((CR⁵R⁶)₁₋₆—CO—NH)₁₋₄—, where R⁵ independently can be H, —CH₃,—(CH₂)₁₋₆CH₃ or —CONH₂ and R⁶ independently can be H, —CH₃,—(CH₂)₁₋₆CH₃; or

a bond

-   -   provided that        -   at least one of Q₁ or Q₂ is not a bond, and        -   that Q₂ is not —CO—(CH₂)₂—CO—NH— when n is 0 or 1, X₁ is a            bond and Q₃ is (CH₂CH₂O)₂—, (CH₂CH₂O)₃— or            (CH₂CH₂OCH₂CH₂CH₂CH₂O)— and        -   that if an amine in Q₁ or Q₂ forms a bond with the rest of            the substituent, the amine must be bound to the rest of the            substituent via a carbonyl group;            Q₃, Q₄, and Q₅ independently of each other can be

—(CH₂)_(m)— where m is an integer in the range of 6 to 32;

a divalent hydrocarbon chain comprising 1, 2 or 3 —CH═CH— groups and anumber of —CH₂— groups sufficient to give a total number of carbon atomsin the chain in the range of 4 to 32;

—CO—((CR⁵R⁶)₁₋₆—NH—CO)—;

—(CO—(CR⁵R⁶)₁₋₆—CO—NH)₁₋₄—, where R⁵ independently can be H, —CH₃,—(CH₂)₁₋₆CH₃ or —CONH₂ and R⁶ independently can be H, —CH₃,—(CH₂)₁₋₆CH₃;

—CO—(CH₂)₀₋₃—Ar—(CH₂)₀₋₃— where Ar can be arylene or heteroarylene,which may be substituted with one or two groups selected from the groupconsisting of —CH₃, —(CH)₁₋₆—CH₃, —CONR¹R² or —SO₂NR¹R² where R¹ and R²independently of each other can be H, —CH₃ or —(CH)₁₋₆-CH₃;

(CH₂CH₂O)_(y)—; (CH₂CH₂CH₂O)_(y)—; (CH₂CH₂CH₂CH₂O)_(y)—;(CH₂CH₂OCH₂CH₂CH₂CH₂O)_(y)— or (CH₂CH₂CH₂OCH₂CH₂CH₂CH₂O)_(y)—;—(CH₂OCH₂)_(y)— where y is 1-20;

arylene or heteroarylene, which may be substituted with one or twogroups selected from the group consisting of —CH₃, —(CH)₁₋₆—CH₃,—CONR¹R² or —SO₂NR¹R² where R¹ and R², independently of each other canbe H, —CH₃ or —(CH)₁₋₆—CH₃; or

a chain of the formula

—(CH₂)_(s)—Y₁—(Ar)_(v1)—Y₂—(CH₂)_(w)—Y₃—(Ar)_(v2)—Y₄—(CH₂)_(t)—Y₅—(Ar)_(v3)—Y₆—(CH₂)_(z)—wherein Ar is defined as above, Y₁-Y₆ independently of each other can beO, S, S═O, SO₂ or a bond; where s, w, t and z independently of eachother are zero or an integer from 1 to 10 so that the sum of s, w, t andz is in the range from 4 to 30, and v₁, v₂, and v₃ independently of eachother can be zero or 1 with the proviso that Y₁-Y₆ do not link to eachother and that the structure —O—(CH₂)₁—O— does not occur; or

a bond;

with the proviso that

-   -   at least one of Q₃-Q₅ is not a bond and    -   Q₂ is not —CO—((CH₂)₁₋₆—NH—CO)₁₋₄— when one of Q₃-Q₅ is arylene        or heteroarylene or a chain of the formula        —(CH₂)_(s)—Y₁—(C₆H₄)_(v1)—Y₂—(CH₂)_(w)—Y₃—(C₆H₄)_(v2)—Y₄—(CH₂)_(t)—Y₅—(C₆H₄)_(v3)—Y₆—(CH₂)_(z)—;        X₁, X₂ and X₃ are independently of each other

O;

—C═O

a bond;

NCOR¹, where R¹ can be H, —CH₃ or —(CH)₁₋₆—CH₃; or

where R is hydrogen, C₁₋₃-alkyl, C₂₋₃-alkenyl or C₂₋₃-alkynyl;

with the proviso that

-   -   —X₁, X₂ and X₃ cannot bind to Z and    -   when X₁, X₂ and X₃ are O then X₁, X₂ and X₃ do not bind directly        to O in Q₃, Q₄, and Q₅        and

Z is:

—COOH;

—CO-Asp;

—CO-Glu;

—CO-Gly;

—CO-Sar;

—CH(COOH)₂;

—N(CH₂COOH)₂;

—SO₃H

—OSO₃H

—OPO3H₂

—PO₃H₂ or

-tetrazol-5-yl or

—O—W₁,

where W₁ is arylene or heteroarylene, which may be substituted with oneor two groups selected from the group consisting of tetrazo-5-lyl,—COOH, —SO₃H, —(CH₂)₁₋₆—SO₃H, —(CH₂)₁₋₆—O—PO₃H₂, —CONR³R⁴ or —SO₂NR³R⁴where R³ and R⁴, independently of each other can be H, —(CH₂)₁₋₆—SO₃H,or —(CH₂)₁₋₆—O—PO₃H₂; provided that when Z is —O—W₁ then Q₁ must bepresent;and any Zn²⁺ complex thereof.

3. An insulin derivative having a formula

wherein Ins is a parent insulin moiety andQ₁-Q₂-[CH₂]_(n)—X₁—[CH₂]_(n)-Q₃-[CH₂]_(n)—X₂—[CH₂]_(n)-Q₄-[CH₂]_(n)—X₃—[CH₂]_(n)-Q₅-[CH₂]_(n)-Zis a substituent and where the Ins is attached to the substituent via anamide bond between the α-amino group of the N-terminal amino acidresidue of the B chain of Ins or an ε-amino group of a Lys residuepresent in the A or B chain of Ins and a CO group in Q₁ or Q₂ of thesubstituent;each n is independently 0, 1, 2, 3, 4, 5 or 6;

Q₁ is:

-   -   an amino acid amide of an amino acid with a carboxylic acid in        the side chain, or an amino acid with an uncharged side chain,        which residue forms, with its carboxylic acid group, an amide        group together with the α-amino group of the N-terminal amino        acid residue of the B chain of Ins or together with the ε-amino        group of a Lys residue present in the A or B chain of Ins, or

a chain composed of two, three or four α-amino acid amide or amino acidresidues as specified above linked together via amide bonds, whichchain—via an amide bond—is linked to the α-amino group of the N-terminalamino acid residue of the B chain of Ins or to the ε-amino group of aLys residue present in the A or B chain of Ins, or

a bond

Q₂ is:

—COCH(CONH₂)—

—COCH₂N(CH₂CONH₂)—

—COCH₂N(CH₂CONH₂)COCH₂N(CH₂CONH₂)

—COCH₂CH₂N(CH₂CH₂CONH₂)—

—COCH₂CH₂N(CH₂CH₂CONH₂)—COCH₂CH₂N(CH₂CH₂CONH₂)—

—COCH₂N(CH₂CH₂CONH₂)—

—COCH₂CH₂N(CH₂CONH₂)—

—COCH₂OCH₂CONH—

—CO—((CR⁵R⁶)₁₋₆—NH—CO)₁₋₄—;

—CO—((CR⁵R⁶)₁₋₆—CO—NH)₁₋₄—, where R⁵ independently can be H, —CH₃,—(CH₂)₁₋₆CH₃ or —CONH₂ and R⁶ independently can be H, —CH₃,—(CH₂)₁₋₆CH₃; or

a bond

-   -   provided that        -   at least one of Q₁ or Q₂ is not a bond, and        -   that Q₂ is not —CO—(CH₂)₂—CO—NH— when n is 0 or 1, X₁ is a            bond and Q₃ is (CH₂CH₂O)₂—, (CH₂CH₂O)₃— or            (CH₂CH₂OCH₂CH₂CH₂CH₂O)— and        -   that if an amine in Q₁ or Q₂ forms a bond with the rest of            the substituent, the amine must be bound to the rest of the            substituent via a carbonyl group;            Q₃, Q₄, and Q₅ independently of each other can be

—(CH₂)_(m)— where m is an integer in the range of 6 to 32;

a divalent hydrocarbon chain comprising 1, 2 or 3 —CH═CH— groups and anumber of —CH₂— groups sufficient to give a total number of carbon atomsin the chain in the range of 4 to 32;

—CO—((CR⁵R⁶)₁₋₆—NH—CO)—;

—(CO—(CR⁵R⁶)₁₋₆—CO—NH)₁₋₄—, where R⁵ independently can be H, —CH₃,—(CH₂)₁₋₆CH₃ or —CONH₂ and R⁶ independently can be H, —CH₃,—(CH₂)₁₋₆CH₃;

—CO—(CH₂)₀₋₃—Ar—(CH₂)₀₋₃— where Ar can be arylene or heteroarylene,which may be substituted with one or two groups selected from the groupconsisting of —CH₃, —(CH)₁₋₆—CH₃, —CONR¹R² or —SO₂NR¹R² where R¹ and R²,independently of each other can be H, —CH₃ or —(CH)₁₋₆—CH₃;

(CH₂CH₂O)_(y)—; (CH₂CH₂CH₂O)_(y)—; (CH₂CH₂CH₂CH₂O)_(y)—;(CH₂CH₂OCH₂CH₂CH₂CH₂O)_(y)— or (CH₂CH₂CH₂OCH₂CH₂CH₂CH₂O)_(y)—;—(CH₂OCH₂)_(y)— where y is 1-20;

arylene or heteroarylene, which may be substituted with one or twogroups selected from the group consisting of —CH₃, —(CH)₁₋₆—CH₃,—CONR¹R² or —SO₂NR¹R², where R¹ and R², independently of each other canbe H, —CH₃ or —(CH)₁₋₆—CH₃; or

a chain of the formula

—(CH₂)_(s)—Y₁—(Ar)_(v1)—Y₂—(CH₂)_(w)—Y₃—(Ar)_(v2)—Y₄—(CH₂)_(t)—Y₅—(Ar)_(v3)—Y₆—(CH₂)_(z)—

wherein Ar is defined as above, Y₁-Y₆ independently of each other can beO, S, S═O, SO₂ or a bond; where s, w, t and z independently of eachother are zero or an integer from 1 to 10 so that the sum of s, w, t andz is in the range from 4 to 30, and v₁, v₂, and v₃ independently of eachother can be zero or 1 with the proviso that Y₁-Y₆ do not link to eachother and that the structure —O—(CH₂)₁—O— does not occur; or

a bond;

with the proviso that

-   -   at least one of Q₃-Q₅ is not a bond and    -   that Q₁ or Q₂ comprises a non-linking amide when one of Q₃-Q₅ is        arylene, heteroarylene or a chain of the formula        —(CH₂)_(s)—Y₁—(C₆H₄)_(v1)—Y₂—(CH₂)_(w)—Y₃—(C₆H₄)_(v2)—Y₄—(CH₂)_(t)—Y₅—(C₆H₄)_(v3)—Y₆—(CH₂)_(z)—.        X₁, X₂ and X₃ are independently of each other

O;

—C═O

a bond;

NCOR¹, where R¹ can be H, —CH₃ or —(CH)₁₋₆—CH₃; or

where R is hydrogen, C₁₋₃-alkyl, C₂₋₃-alkenyl or C₂₋₃-alkynyl;

with the proviso that

-   -   X₁, X₂ and X₃ cannot bind to Z and    -   when X₁, X₂ and X₃ are O then X₁, X₂ and X₃ do not bind directly        to O in Q₃, Q₄, and Q₅        and

Z is:

—COOH;

—CO-Asp;

—CO-Glu;

—CO-Gly;

—CO-Sar;

—CH(COOH)₂;

—N(CH₂COOH)₂;

—SO₃H

—OSO₃H

—OPO3H₂

—PO₃H₂ or

-tetrazol-5-yl or

—O—W₁,

where W₁ is arylene or heteroarylene, which may be substituted with oneor two groups selected from the group consisting of tetrazo-5-lyl,—COOH, —SO₃H, —(CH₂)₁₋₆—SO₃H, —(CH₂)₁₋₆—O—PO₃H₂, —CONR³R⁴ or —SO₂NR³R⁴where R³ and R⁴ independently of each other can be H, —(CH₂)₁₋₆—SO₃H, or—(CH₂)₁₋₆—O—PO₃H₂; provided that when Z is —O—W₁ then Q₁ must be presentand any Zn²⁺ complex thereof.

4. An insulin derivative having a formula

wherein Ins is a parent insulin moiety andQ₁-Q₂-[CH₂]_(n)—X₁—[CH₂]_(n)-Q₃-[CH₂]_(n)—X₂—[CH₂]_(n)-Q₄-[CH₂]_(n)—X₃—[CH₂]_(n)-Q₅-[CH₂]_(n)-Zis a substituent and where the Ins is attached to the substituent via anamide bond between the α-amino group of the N-terminal amino acidresidue of the B chain of Ins or an ε-amino group of a Lys residuepresent in the A or B chain of Ins and a CO group in Q₁ or Q₂ of thesubstituent;each n is independently 0, 1, 2, 3, 4, 5 or 6;

Q₁ is:

-   -   an amino acid amide of an amino acid with a carboxylic acid in        the side chain, or an amino acid with an uncharged side chain,        which residue forms, with its carboxylic acid group, an amide        group together with the α-amino group of the N-terminal amino        acid residue of the B chain of Ins or together with the ε-amino        group of a Lys residue present in the A or B chain of Ins, or

a chain composed of two, three or four α-amino acid amide or amino acidresidues as specified above linked together via amide bonds, whichchain—via an amide bond—is linked to the α-amino group of the N-terminalamino acid residue of the B chain of Ins or to the ε-amino group of aLys residue present in the A or B chain of Ins, or

a bond

Q₂ is:

—COCH(CONH₂)—

—COCH₂N(CH₂CONH₂)—

—COCH₂N(CH₂CONH₂)COCH₂N(CH₂CONH₂)

—COCH₂CH₂N(CH₂CH₂CONH₂)—

—COCH₂CH₂N(CH₂CH₂CONH₂)—COCH₂CH₂N(CH₂CH₂CONH₂)—

—COCH₂N(CH₂CH₂CONH₂)—

—COCH₂CH₂N(CH₂CONH₂)—

—COCH₂OCH₂CONH—

—CO—((CR⁵R⁶)₁₋₆—CO—NH)₁₋₄—, where R⁵ independently can be H, —CH₃,—(CH₂)₁₋₆CH₃ or —CONH₂ and R⁶ independently can be H, —CH₃,—(CH₂)₁₋₆CH₃; or

a bond

-   -   provided that        -   at least one of Q₁ or Q₂ is not a bond, and        -   that Q₂ is not —CO—(CH₂)₂—CO—NH— when n is 0 or 1, X₁ is a            bond and Q₃ is (CH₂CH₂O)₂—, (CH₂CH₂O)₃— or            (CH₂CH₂OCH₂CH₂CH₂CH₂O)— and        -   that if an amine in Q₁ or Q₂ forms a bond with the rest of            the substituent, the amine must be bound to the rest of the            substituent via a carbonyl group;            -   Q₃, Q₄, and Q₅ independently of each other can be

—(CH₂)_(m)— where m is an integer in the range of 6 to 32;

a divalent hydrocarbon chain comprising 1, 2 or 3 —CH═CH— groups and anumber of —CH₂— groups sufficient to give a total number of carbon atomsin the chain in the range of 4 to 32;

—CO—((CR⁵R⁶)₁₋₆—NH—CO)—;

—(CO—(CR⁵R⁶)₁₋₆—CO—NH)₁₋₄—, where R⁵ independently can be H, —CH₃,—(CH₂)₁₋₆CH₃ or —CONH₂ and R⁶ independently can be H, —CH₃,—(CH₂)₁₋₆CH₃;

—CO—(CH₂)₀₋₃—Ar—(CH₂)₀₋₃— where Ar can be arylene or heteroarylene,which may be substituted with one or two groups selected from the groupconsisting of —CH₃, —(CH)₁₋₆—CH₃, —CONR¹R² or —SO₂NR¹R² where R¹ and R²,independently of each other can be H, —CH₃ or —(CH)₁₋₆—CH₃;

(CH₂CH₂O)_(y)—; (CH₂CH₂CH₂O)_(y)—; (CH₂CH₂CH₂CH₂O)_(y)—;(CH₂CH₂OCH₂CH₂CH₂CH₂O)_(y)— or (CH₂CH₂CH₂OCH₂CH₂CH₂CH₂O)_(y)—;—(CH₂OCH₂)_(y)— where y is 1-20;

-   -   arylene or heteroarylene, which may be substituted with one or        two groups selected from the group consisting of —CH₃,        —(CH)₁₋₆—CH₃, —CONR¹R² or —SO₂NR¹R² where R¹ and R²,        independently of each other can be H, —CH₃ or —(CH)₁₋₆—CH₃; or

a chain of the formula

—(CH₂)_(s)—Y₁—(Ar)_(v1)-Y₂-(CH₂)_(w)—Y₃—(Ar)_(v2)—Y₄—(CH₂)_(t)—Y₅—(Ar)_(v3)—Y₆—(CH₂)_(z)—

wherein Ar is defined as above, Y₁-Y₆ independently of each other can beO, S, S═O, SO₂ or a bond; where s, w, t and z independently of eachother are zero or an integer from 1 to 10 so that the sum of s, w, t andz is in the range from 4 to 30, and v₁, v₂, and v₃ independently of eachother can be zero or 1 with the proviso that Y₁-Y₆ do not link to eachother and that the structure —O—(CH₂)₁—O— does not occur; or

a bond;

with the proviso that at least one of Q₃-Q₅ is not a bond;X₁, X₂ and X₃ are independently of each other

O;

—C═O

a bond;

NCOR¹, where R¹ can be H, —CH₃ or —(CH)₁₋₆—CH₃; or

where R is hydrogen, C₁₋₃-alkyl, C₂₋₃-alkenyl or C₂₋₃-alkynyl;

with the proviso that

-   -   —X₁, X₂ and X₃ cannot bind to Z and    -   when X₁, X₂ and X₃ are O then X₁, X₂ and X₃ do not bind directly        to O in Q₃, Q₄, and Q₅        and

Z is:

—COOH;

—CO-Asp;

—CO-Glu;

—CO-Gly;

—CO-Sar;

—CH(COOH)₂;

—N(CH₂COOH)₂;

—SO₃H

OSO₃H

—OPO3H₂

—PO₃H₂ or

-tetrazol-5-yl or

—O—W₁,

where W₁ is arylene or heteroarylene, which may be substituted with oneor two groups selected from the group consisting of tetrazo-5-lyl,—COOH, —SO₃H, —(CH₂)₁₋₆—SO₃H, —(CH₂)₁₋₆—O—PO₃H₂, —CONR³R⁴ or —SO₂NR³R⁴where R³ and R⁴, independently of each other can be H, —(CH₂)₁₋₆—SO₃H,or —(CH₂)₁₋₆—O—PO₃H₂; provided that when Z is —O—W₁ then Q₁ must bepresentand any Zn²⁺ complex thereof.

5. Insulin derivative according to paragraph 1-4, wherein Q₁ is an aminoacid amide of an amino acid with a carboxylic acid in the side chain, oran amino acid with an uncharged side chain, the amino acid amide residueor amino acid residue having from 2 to 10 carbon atoms.

6. Insulin derivative according to any of paragraphs 1-5, wherein Q₁ isselected from the group consisting of β-D-Asp-amide, β-L-Asp-amide,γ-L-Glu-amide and γ-D-Glu-amide.

7. Insulin derivative according to paragraphs 1-4, wherein Q₁ is a chaincomposed of two, three or four α-amino acid amide residues or amino acidresidues with an uncharged side chain.

8. Insulin derivative according to paragraphs 1-4 or 7, wherein Q₁ is achain of two amino acid amide residues selected from the groupconsisting of β-L-Asp-amide-β-L-Asp-amide, β-L-Asp-amide-γ-L-Glu-amide,γ-L-Glu-amide-γ-L-Glu-amide, γ-L-Glu-amide-β-L-Asp-amide,β-L-Asp-amide-β-D-Asp-amide, β-L-Asp-amide-γ-D-Glu-amide,γ-L-Glu-amide-γ-D-Glu-amide, γ-L-Glu-amide-β-D-Asp-amide,β-D-Asp-amide-β-L-Asp-amide, β-D-Asp-amide-γ-L-Glu-amide,γ-D-Glu-amide-γ-L-Glu-amide, γ-D-Glu-amide-β-L-Asp-amide,β-D-Asp-amide-β-D-Asp-amide, β-D-Asp-amide-γ-D-Glu-amide,γ-D-Glu-amide-γ-D-Glu-amide, γ-D-Glu-amide-β-D-Asp-amide.

9. Insulin derivative according to any of paragraphs 1-8, wherein Q₂ isa bond

10. Insulin derivative according to any of paragraphs 1-3 or 5-8,wherein Q₂ is —CO—((CR⁵R⁶)₁₋₆—NH—CO)₁₋₄—.

11. Insulin derivative according to paragraph 10, where Q₂ is selectedfrom the group consisting of —CO—((CH₂)₂—NH—CO)₁—, —CO—((CH₂)₃—NH—CO)₁—and —CO—((CH₂)₄—NH—CO)₁—, —CO—((CH₂)₅—NH—CO)₁—.

12. Insulin derivative according to paragraphs 1, 3 or 10-11, wherein Q₁is a bond.

13. Insulin derivative according to paragraphs 1-3 wherein

Q₁ is:

an amino acid amide of an amino acid with a carboxylic acid in the sidechain, or an amino acid with an uncharged side chain, which residueforms, with its carboxylic acid group, an amide group together with theα-amino group of the N-terminal amino acid residue of the B chain of Insor together with the ε-amino group of a Lys residue present in the A orB chain of Ins, or

a chain composed of two, three or four α-amino acid amide or amino acidresidues as specified above linked together via amide bonds, in whichthe chain—via an amide bond—is linked to the α-amino group of theN-terminal amino acid residue of the B chain of Ins or to the ε-aminogroup of a Lys residue present in the A or B chain of Ins, or

a bond

Q₂ is:

—COCH(CONH₂)—

—COCH₂N(CH₂CONH₂)—

—COCH₂N(CH₂CONH₂)COCH₂N(CH₂CONH₂)

—COCH₂CH₂N(CH₂CH₂CONH₂)—

—COCH₂CH₂N(CH₂CH₂CONH₂)—COCH₂CH₂N(CH₂CH₂CONH₂)—

—COCH₂N(CH₂CH₂CONH₂)—

—COCH₂CH₂N(CH₂CONH₂)—

—COCH₂OCH₂CONH—

—CO—((CR⁵R⁶)₁₋₆—NH—CO)₁₋₄—;

—(CO—(CR⁵R⁶)₁₋₆—CO—NH)₁₋₄—, where R⁵ can be H, —CH₃, —(CH₂)₁₋₆CH₃ or—CONH₂ and R⁶ can be H, —CH₃, —(CH₂)₁₋₆CH₃; or

a bond

provided that at least one of Q₁ or Q₂ is not a bond and that if anamine in Q₁ or Q₂ forms a bond with the rest of the substituent, theamine must be bound to the rest of the substituent via a carbonyl group;

Q₃ is

—(CH₂)_(m)— where m is an integer in the range of 6 to 32;

a divalent hydrocarbon chain comprising 1, 2 or 3 —CH═CH— groups and anumber of —CH₂— groups sufficient to give a total number of carbon atomsin the chain in the range of 4 to 32; or

a divalent hydrocarbon chain of the formula —(CH₂)_(s)C₆H₄(CH₂)_(w)—wherein v and w are integers or one of them is zero so that the sum of sand w is in the range of 6 to 30;

X₁, can be —C═O or a bond;Q₄, Q₅, X₂ and X₃ are bonds;All values of n are zero; and

Z is:

—COOH;

—CO-Asp;

—CO-Glu;

—CO-Gly;

—CO-Sar;

—CH(COOH)₂;

—N(CH₂COOH)₂;

—SO₃H

—OSO₃H

—OPO3H₂

—PO₃H₂ or

-tetrazol-5-yl

and any Zn²⁺ complex thereof

14. Insulin derivative according to paragraph 13, wherein Q₁ is an aminoacid amide of an amino acid with a carboxylic acid in the side chain, oran amino acid with an uncharged side chain, the amino acid amide residueor amino acid residue having from 2 to 10 carbon atoms.

15. Insulin derivative according to any of paragraphs 13-14, wherein Q₁is selected from the group consisting of β-D-Asp-amide, β-L-Asp-amide,γ-L-Glu-amide and γ-D-Glu-amide.

16. Insulin derivative according to paragraph 13, wherein Q₁ is a chaincomposed of two, three or four α-amino acid amide residues and/or aminoacid residues with an uncharged side chain.

17. Insulin derivative according to paragraphs 13 or 16, wherein Q₁ is achain of two amino acid amide residues selected from the groupconsisting of β-L-Asp-amide-β-L-Asp-amide, β-L-Asp-amide-γ-L-Glu-amide,γ-L-Glu-amide-γ-L-Glu-amide, γ-L-Glu-amide-β-L-Asp-amide,β-L-Asp-amide-β-D-Asp-amide, β-L-Asp-amide-γ-D-Glu-amide,γ-L-Glu-amide-γ-D-Glu-amide, γ-L-Glu-amide-β-D-Asp-amide,β-D-Asp-amide-β-L-Asp-amide, β-D-Asp-amide-γ-L-Glu-amide,γ-D-Glu-amide-γ-L-Glu-amide, γ-D-Glu-amide-β-L-Asp-amide,β-D-Asp-amide-β-D-Asp-amide, β-D-Asp-amide-γ-D-Glu-amide,γ-D-Glu-amide-γ-D-Glu-amide, γ-D-Glu-amide-β-D-Asp-amide.

18. Insulin derivative according to any of paragraphs 13-17, wherein Q₂is a bond.

19. Insulin derivative according to paragraphs 13-17, wherein Q₂ isselected from the group consisting of

—COCH(CONH₂)—

—COCH₂N(CH₂CONH₂)—

—COCH₂N(CH₂CONH₂)COCH₂N(CH₂CONH₂)

—COCH₂CH₂N(CH₂CH₂CONH₂)—

—COCH₂CH₂N(CH₂CH₂CONH₂)—COCH₂CH₂N(CH₂CH₂CONH₂)—

—COCH₂N(CH₂CH₂CONH₂)—

—COCH₂CH₂N(CH₂CONH₂)—

—COCH₂OCH₂CONH—;

—CO—((CR⁵R⁶)₁₋₆—NH—CO)₁₋₄—; or

—CO—((CR⁵R⁶)₁₋₆—CO—NH)₁₋₄—, where R⁵ independently can be H, —CH₃,—(CH₂)₁₋₆CH₃ or —CONH₂ and R⁶ independently can be H, —CH₃, —(CH₂)₁₋₆CH₃

20. Insulin derivative according to paragraph 19, wherein Q₂ is selectedfrom the group consisting of —COCH₂OCH₂CONH—, —CO—((CH₂)₂—NH—CO)₁—,—CO—((CH₂)₃—NH—CO)₁—, —CO—((CH₂)₄—NH—CO)₁— and —CO—((CH₂)₅—NH—CO)₁—.

21. Insulin derivative according to paragraphs 13 or 19-21, wherein Q₁is a bond.

22. Insulin derivative according to any of paragraphs 13-21, wherein X₁is —C═O.

23. Insulin derivative according to any of paragraphs 13-22, wherein Q₃is —(CH₂)_(m)— where m is an integer in the range of 6 to 32 or from 8to 20.

24. Insulin derivative according to paragraph 23, where m is 12, 13, 14,15 or 16.

25. Insulin derivative according to paragraphs 13-22, wherein Q₃ is adivalent hydrocarbon chain comprising 1, 2 or 3 —CH═CH— groups and anumber of —CH₂— groups sufficient to give a total number of carbon atomsin the chain in the range of 4 to 32.

26. Insulin derivative according to any of paragraphs 13-25, wherein Zis —COOH.

27. Insulin derivative according to any of paragraphs 13-25, wherein Zis —CH(COOH)₂.

28. An insulin derivative according to any of paragraphs 13-25, whereinZ is —N(CH₂COOH)₂.

29. An insulin derivative according to any of paragraphs 13-25, whereinZ is —SO₃H.

30. An insulin derivative according to any of paragraphs 13-25, whereinZ is —PO₃H.

31. Insulin derivative according to any of paragraphs 13-30 wherein theinsulin derivative is selected from the group consisting of

-   N^(εB29)-ω-carboxy-pentadecanoyl-γ-L-glutamylamide desB30 human    insulin,-   N^(εB29)-ω-carboxy-pentadecanoyl-γ-amino-butanoyl desB30 human    insulin,-   N^(εB29)-ω-carboxy-tetradecanoyl-γ-L-glutamylamide desB30 human    insulin,-   N^(εB29)-ω-carboxy-tridecanoyl-γ-L-glutamylamide desB30 human    insulin,-   N^(εB29)-ω-carboxy-pentadecanoyl-β-alanyl desB30 human insulin,-   N^(εB29)-ω-carboxy-pentadecanoyl-γ-L-aspartylamide desB30 human    insulin,-   N^(εB29)-ω-carboxy-pentadecanoyl-ε-aminohexanoyl desB30 human    insulin,-   N^(εB29)-ω-carboxy-pentadecanoyl-δ-aminopentanoyl desB30 human    insulin,-   N^(εB29)-ω-carboxy-tridecanoyl-γ-amino-butanoyl desB30 human insulin-   N^(εB29)-ω-carboxy-undecanoyl-δ-amino-butanoyl desB30 human insulin    and-   N^(εB29)-ω-carboxy-tetradecanoyl-γ-amino-butanoyl desB30 human    insulin

32. Insulin derivative according to paragraph 2 or 4, wherein

Q₃ is:

—CO—((CR⁵R⁶)₁₋₆—NH—CO)—;

—(CO—(CR⁵R⁶)₁₋₆—CO—NH)₁₋₄—, where R⁵ independently can be H, —CH₃,—(CH₂)₁₋₆CH₃ or —CONH₂ and R⁶ independently can be H, —CH₃,—(CH₂)₁₋₆CH₃;

—CO—(CH₂)₀₋₃—Ar—(CH₂)₀₋₃— where Ar can be arylene or heteroarylene,which may be substituted with one or two groups selected from the groupconsisting of —CH₃, —(CH)₁₋₆—CH₃, —CONR¹R² or —SO₂NR¹R² where R¹ and R²independently of each other can be H, —CH₃ or —(CH)₁₋₆—CH₃; or

a bond

Q₄ is:

—(CH₂)_(m)— where m is an integer from 4 to 22;

a divalent hydrocarbon chain comprising 1, 2 or 3 —CH═CH— groups and anumber of —CH₂— groups sufficient to give a total number of carbon atomsin the chain in the range of 4 to 22;

-   -   arylene or heteroarylene, which may be substituted with one or        two groups selected from the group consisting of —CH₃,        —(CH)₁₋₆—CH₃, —CONR¹R² or —SO₂NR¹R², where R¹ and R²,        independently of each other can be H, —CH₃ or —(CH)₁₋₆—CH₃; or    -   a chain of the formula

—(CH₂)_(s)—Y₁—(Ar)_(v1)—Y₂—(CH₂)_(w)—Y₃—(Ar)_(v2)—Y₄—(CH₂)_(t)—Y₅—(Ar)_(v3)—Y₆—(CH₂)_(z)—

wherein Ar is defined as above, Y₁-Y₆ independently of each other can beO, S, S═O, SO₂ or a bond; where s, w, t and z independently of eachother are zero or an integer from 1 to 10 so that the sum of s, w, t andz is in the range from 4 to 30, and v₁, v₂, and v₃ independently of eachother can be zero or 1 with the proviso that Y₁-Y₆ do not link to eachother and that the structure —O—(CH₂)₁—O— does not occur;

X₁ is:

O;

—C═O

NCOR¹, where R¹ can be H, —CH₃ or —(CH)₁₋₆—CH₃; or

-   -   where R is hydrogen, C₁₋₃-alkyl, C₂₋₃-alkenyl or C₂₋₃-alkynyl;    -   with the proviso that when X₁ is O, then X₁ does not bind        directly to O in Q₄;        X₂, X₃ and Q₅ are bonds;        All values of n are zero; and

Z is:

—COOH;

—CO-Asp;

—CO-Glu;

—CO-Gly;

—CO-Sar;

—CH(COOH)₂;

—N(CH₂COOH)₂;

—SO₃H

—OSO₃H

—OPO3H₂

—PO₃H₂ or

-tetrazol-5-yl or

—O—W₁,

where W₁ is arylene or heteroarylene, which may be substituted with oneor two groups selected from the group consisting of tetrazo-5-lyl,—COOH, —SO₃H, —(CH₂)₁₋₆—SO₃H, —(CH₂)₁₋₆—O—PO₃H₂, —CONR³R⁴ or —SO₂NR³R⁴,where R³ and R⁴ independently of each other can be H, —(CH₂)₁₋₆—SO₃H, or—(CH₂)₁₋₆—O—PO₃H₂; and any Zn²⁺ complex thereof.

33. Insulin derivative according to paragraphs 2, 4 or 32, wherein Q₁ isan amino acid amide of an amino acid with a carboxylic acid in the sidechain, or an amino acid with an uncharged side chain, the amino acidamide residue or amino acid residue having from 2 to 10 carbon atoms.

34. Insulin derivative according to paragraphs 2, 4 or 33, wherein Q₁ isselected from the group consisting of β-D-Asp-amide, β-L-Asp-amide,γ-L-Glu-amide and γ-D-Glu-amide.

35. Insulin derivative according to paragraph 2, 4 or 34, wherein Q₁ isa chain composed of two, three or four α-amino acid amide residues oramino acid residues with an uncharged side chain.

36. Insulin derivative according to paragraphs 2, 4 or 35, wherein Q₁ isa chain of two amino acid amide residues selected from the groupconsisting of β-L-Asp-amide-β-L-Asp-amide, β-L-Asp-amide-γ-L-Glu-amide,γ-L-Glu-amide-γ-L-Glu-amide, γ-L-Glu-amide-β-L-Asp-amide,β-L-Asp-amide-β-D-Asp-amide, β-L-Asp-amide-γ-D-Glu-amide,γ-L-Glu-amide-γ-D-Glu-amide, γ-L-Glu-amide-β-D-Asp-amide,β-D-Asp-amide-β-L-Asp-amide, β-D-Asp-amide-γ-L-Glu-amide,γ-D-Glu-amide-γ-L-Glu-amide, γ-D-Glu-amide-β-L-Asp-amide,β-D-Asp-amide-β-D-Asp-amide, β-D-Asp-amide-γ-D-Glu-amide,γ-D-Glu-amide-γ-D-Glu-amide, γ-D-Glu-amide-β-D-Asp-amide.

37. Insulin derivative according to any of paragraphs 2, 4 or 32-36,wherein Q₂ is a bond.

38. Insulin derivative according to paragraphs 2, 4 or 32-36, where Q₂is —CO—CH₂—(CHCONH₂)—NH—CO; —CO—(CH₂)₂—(CHCONH₂)—NH—CO)— or—CO—(CH₂)₃—(CHCONH₂)—NH—CO)—.

39. Insulin derivative according to paragraph 2, 4 or 38, wherein Q₁ isa bond.

40. Insulin derivative according to paragraphs 32-39, wherein Q₃ is—(CH₂)_(s)—Y₁—(Ar)_(v1)—Y₂—(CH₂)_(w)—Y₃—(Ar)_(v2)—Y₄—(CH₂)_(t)—Y₅—(Ar)_(v3)—Y₆—(CH₂)_(z)—

wherein Ar is defined as above, Y₁-Y₆ independently of each other can beO, S, S═O, SO₂ or a bond; where s, w, t and z independently of eachother are zero or an integer from 1 to 10 so that the sum of s, w, t andz is in the range from 4 to 30, and v₁, v₂, and v₃ independently of eachother can be zero or 1 with the proviso that Y₁-Y₆ do not link to eachother and that the structure —O—(CH₂)₁—O— does not occur.

41. Insulin derivative according to any of paragraph 40, wherein atleast two of v₁, v₂, or v₃ are zero.

42. Insulin derivative according to any of paragraphs 40-41, whereinY₁-Y₆ are bonds.

43. Insulin derivative according to any of paragraphs 40-41, wherein atleast one of Y₁-Y₆ are O or S.

44. Insulin derivative according to any of paragraphs 40-41 or 43,wherein Y₁ is O or S and v₁ is one.

45. Insulin derivative according to any of paragraphs 40-44, wherein sis 6, 7, 8, 9 or 10.

46. Insulin derivative according to any of paragraphs 40-45, wherein X₁,X₂, X₃, Q₄, and Q₅ are bonds and Ar is C₆H₄.

47. Insulin derivative according to any of paragraphs 32-46, wherein nis zero.

48. Insulin derivative according to any of paragraphs 32-47, wherein Zis —COOH.

49. Insulin derivative according to any of paragraphs 32-47, wherein Zis —CH(COOH)₂.

50. An insulin derivative according to any of paragraphs 32-47, whereinZ is —N(CH₂COOH)₂.

51. An insulin derivative according to any of paragraphs 32-47, whereinZ is —SO₃H.

52. An insulin derivative according to any of paragraphs 32-47, whereinZ is —PO₃H.

53. An insulin derivative according to any of paragraphs 32-47, whereinZ is —O—W₁, where W₁ is arylene or heteroarylene, which may besubstituted with one or two groups selected from the group consisting oftetrazo-5-lyl, —COOH, —SO₃H, —(CH₂)₁₋₆—SO₃H, —(CH₂)₁₋₆—O—PO₃H₂,—CONR¹³R¹⁴ or —SO₂NR¹³R¹⁴ where R¹³ and R¹⁴, independently of each othercan be H, —(CH₂)₁₋₆—SO₃H, or —(CH₂)₁₋₆—O—PO₃H₂.

54. Insulin derivative according to any of paragraphs 32-53 wherein theinsulin derivative isN^(εB29)-10-(4-carboxyphenoxy)-decanoyl-γ-L-glutamylamide desB30 humaninsulin.

55. Insulin derivative according to paragraph 1, wherein Q₁ is an aminoacid amide of an amino acid with a carboxylic acid in the side chain, oran amino acid with an uncharged side chain, the amino acid amide residueor amino acid residue having from 2 to 10 carbon atoms.

56. Insulin derivative according to any of paragraphs 1 or 55, whereinQ₁ is selected from the group consisting of β-D-Asp-amide,β-L-Asp-amide, γ-L-Glu-amide and γ-D-Glu-amide.

57. Insulin derivative according to paragraph 1, wherein Q₁ is a chaincomposed of two, three or four α-amino acid amide residues or amino acidresidues with an uncharged side chain.

58. Insulin derivative according to paragraphs 1 or 57, wherein Q₁ is achain of two amino acid amide residues selected from the groupconsisting of β-L-Asp-amide-β-L-Asp-amide, β-L-Asp-amide-γ-L-Glu-amide,γ-L-Glu-amide-γ-L-Glu-amide, γ-L-Glu-amide-β-L-Asp-amide,β-L-Asp-amide-β-D-Asp-amide, β-L-Asp-amide-γ-D-Glu-amide,γ-L-Glu-amide-γ-D-Glu-amide, γ-L-Glu-amide-β-D-Asp-amide,β-D-Asp-amide-β-L-Asp-amide, β-D-Asp-amide-γ-L-Glu-amide,γ-D-Glu-amide-γ-L-Glu-amide, γ-D-Glu-amide-β-L-Asp-amide,β-D-Asp-amide-β-D-Asp-amide, β-D-Asp-amide-γ-D-Glu-amide,γ-D-Glu-amide-γ-D-Glu-amide, γ-D-Glu-amide-β-D-Asp-amide.

59. Insulin derivative according to any of paragraphs 1 or 55-58,wherein Q₂ is a bond.

60. Insulin derivative according to any of paragraphs 1 or 55-58,wherein Q₂ is selected from the group consisting of—CO—(CR⁵R⁶)₁₋₆—NH—CO— and —(CO—(CR⁵R⁶)₁₋₆—CO—NH)₁₋₄—, where R⁵independently can be H, —CH₃, —(CH₂)₁₋₆CH₃ or —CONH₂ and R⁶independently can be H, —CH₃, —(CH₂)₁₋₆CH₃;

61. Insulin derivative according to paragraphs 1 or 60, where Q₂ is—CO—((CH₂)₃—NH—CO)—, —CO—CH₂—(CHCONH₂)—NH—CO;—CO—(CH₂)₂—(CHCONH₂)—NH—CO)— or —CO—(CH₂)₃—(CHCONH₂)—NH—CO)—

62. Insulin derivative according to paragraphs 1 or 60-61, wherein Q₁ isa bond.

63. Insulin derivative according to paragraph 55-62, wherein Q₃ is—(CH₂)_(s)—Y₁—(Ar)_(v1)—Y₂—(CH₂)_(w)—Y₃—(Ar)_(v2)—Y₄—(CH₂)_(t)—Y₅—(Ar)_(v3)—Y₆—(CH₂)_(z)—

wherein Ar is defined as above, Y₁-Y₆ independently of each other can beO, S, S═O, SO₂ or a bond; where s, w, t and z independently of eachother are zero or an integer from 1 to 10 so that the sum of s, w, t andz is in the range from 4 to 30, and v₁, v₂, and v₃ independently of eachother can be zero or 1 with the proviso that Y₁-Y₆ do not link to eachother and that the structure —O—(CH₂)₁—O— does not occur.

64. Insulin derivative according to paragraph 63, wherein at least twoof v₁, v₂, or v₃ are zero.

65. Insulin derivative according to any of paragraphs 63-64, whereinY₁-Y₆ are bonds.

66. Insulin derivative according to any of paragraphs 63-64, wherein atleast one of Y₁—Y₆ are O or S.

67. Insulin derivative according to any of paragraphs 63-64 or 66,wherein Y₁ is O or S and v₁ is one.

68. Insulin derivative according to any of paragraphs 63-67, wherein sis 6, 7, 8, 9 or 10.

69. Insulin derivative according to any of paragraphs 55-68, wherein X₁,X₂, X₃, Q₄, and Q₅ are bonds and Ar is C₆H₄.

70. Insulin derivative according to any of paragraphs 55-69, wherein nis zero.

71. Insulin derivative according to any of paragraphs 55-70, wherein Zis —COOH.

72. Insulin derivative according to any of paragraphs 55-70, wherein Zis —CH(COOH)₂.

73. An insulin derivative according to any of paragraphs 55-70, whereinZ is —N(CH₂COOH)₂.

74. An insulin derivative according to any of paragraphs 55-70, whereinZ is —SO₃H.

75. An insulin derivative according to any of paragraphs 55-70, whereinZ is —PO₃H.

76. An insulin derivative according to any of paragraphs 55-70, whereinZ is —O—W₁, where W₁ is arylene or heteroarylene, which may besubstituted with one or two groups selected from the group consisting oftetrazo-5-lyl, —COOH, —SO₃H, —(CH₂)₁₋₆—SO₃H, —(CH₂)₁₋₆—O—PO₃H₂,—CONR¹³R¹⁴ or —SO₂NR¹³R¹⁴ where R¹³ and R¹⁴, independently of each othercan be H, —(CH₂)₁₋₆—SO₃H, or —(CH₂)₁₋₆—O—PO₃H₂.

77. Insulin derivative according to any of paragraphs 1 or 55-70 whereinthe insulin derivative are selected from the group consisting ofN^(εB29)-10-(4-carboxyphenoxy)-decanoyl-γ-L-glutamylamide desB30 humaninsulin, N^(εB29)-4-[11-(4-Carboxyphenyl)undecanoylamino]butyryl desB30human insulin.

78. Insulin derivative according to paragraph 3, wherein Q₁ is an aminoacid amide of an amino acid with a carboxylic acid in the side chainhaving from 2 to 10 carbon atoms.

79. Insulin derivative according to any of paragraphs 3 or 77, whereinQ₁ is selected from the group consisting of β-D-Asp-amide,β-L-Asp-amide, γ-L-Glu-amide and γ-D-Glu-amide.

80. Insulin derivative according to paragraphs 3, wherein Q₁ is a chainof two amino acid amide residues selected from the group consisting ofβ-L-Asp-amide-β-L-Asp-amide, β-L-Asp-amide-γ-L-Glu-amide,γ-L-Glu-amide-γ-L-Glu-amide, γ-L-Glu-amide-β-L-Asp-amide,β-L-Asp-amide-β-D-Asp-amide, β-L-Asp-amide-γ-D-Glu-amide,γ-L-Glu-amide-γ-D-Glu-amide, γ-L-Glu-amide-β-D-Asp-amide,β-D-Asp-amide-β-L-Asp-amide, β-D-Asp-amide-γ-L-Glu-amide,γ-D-Glu-amide-γ-L-Glu-amide, γ-D-Glu-amide-β-L-Asp-amide,β-D-Asp-amide-β-D-Asp-amide, β-D-Asp-amide-γ-D-Glu-amide,γ-D-Glu-amide-γ-D-Glu-amide, γ-D-Glu-amide-β-D-Asp-amide.

81. Insulin derivative according to any of paragraphs 3 or 77-79,wherein Q₂ is a bond.

82. Insulin derivative according to paragraphs 3 or 77-80, wherein Q₂ isselected from the group consisting of

—COCH(CONH₂)—

—COCH₂N(CH₂CONH₂)—

—COCH₂N(CH₂CONH₂)COCH₂N(CH₂CONH₂)

—COCH₂CH₂N(CH₂CH₂CONH₂)—

—COCH₂CH₂N(CH₂CH₂CONH₂)—COCH₂CH₂N(CH₂CH₂CONH₂)—

—COCH₂N(CH₂CH₂CONH₂)—

—COCH₂CH₂N(CH₂CONH₂)—

—COCH₂OCH₂CONH—;

—CO—((CR⁵R⁶)₁₋₆—NH—CO)₁₋₄—; or

—CO—((CR⁵R⁶)₁₋₆—CO—NH)₁₋₄—, where R⁵ independently can be H, —CH₃,—(CH₂)₁₋₆CH₃ or —CONH₂ and R⁶ independently can be H, —CH₃, —(CH₂)₁₋₆CH₃provided that Q₂ comprises a non-linking amide if Q₁ is a bond.

83. Insulin derivative according to paragraphs 3 or 82, wherein Q₁ is abond.

84. Insulin derivative according to paragraph 78-83, wherein Q₃ is—(CH₂)_(s)—Y₁—(Ar)_(v1)—Y₂—(CH₂)_(w)—Y₃—(Ar)_(v2)—Y₄—(CH₂)_(t)—Y₅—(Ar)_(v3)—Y₆—(CH₂)_(z)—

wherein Ar is defined as above, Y₁-Y₆ independently of each other can beO, S, S═O, SO₂ or a bond; where s, w, t and z independently of eachother are zero or an integer from 1 to 10 so that the sum of s, w, t andz is in the range from 4 to 30, and v₁, v₂, and v₃ independently of eachother can be zero or 1 with the proviso that Y₁-Y₆ do not link to eachother and that the structure —O—(CH₂)₁—O— does not occur.

85. Insulin derivative according to paragraph 84, wherein at least twoof v₁, v₂, or v₃ are zero.

86. Insulin derivative according to any of paragraphs 84-85, whereinY₁-Y₆ are bonds.

87. Insulin derivative according to any of paragraphs 84-85, wherein atleast one of Y₁—Y₆ are O or S.

88. Insulin derivative according to any of paragraphs 84-85 or 87,wherein Y₁ is O or S and v₁ is one.

89. Insulin derivative according to any of paragraphs 84-88, wherein sis 6, 7, 8, 9 or 10.

90. Insulin derivative according to any of paragraphs 78-89, wherein X₁,X₂, X₃, Q₄, and Q₅ are bonds and Ar is C₆H₄.

91. Insulin derivative according to any of paragraphs 78-90, wherein nis zero.

92. Insulin derivative according to any of paragraphs 78-91, wherein Zis —COOH.

93. Insulin derivative according to any of paragraphs 78-91, wherein Zis —CH(COOH)₂.

94. An insulin derivative according to any of paragraphs 78-91, whereinZ is —N(CH₂COOH)₂.

95. An insulin derivative according to any of paragraphs 78-91, whereinZ is —SO₃H.

96. An insulin derivative according to any of paragraphs 78-91, whereinZ is —PO₃H.

97. An insulin derivative according to any of paragraphs 78-91, whereinZ is —O—W₁, where W₁ is arylene or heteroarylene, which may besubstituted with one or two groups selected from the group consisting oftetrazo-5-lyl, —COOH, —SO₃H, —(CH₂)₁₋₆—SO₃H, —(CH₂)₁₋₆—O—PO₃H₂,—CONR¹³R¹⁴ or —SO₂NR¹³R¹⁴ where R¹³ and R¹⁴ independently of each othercan be H, —(CH₂)₁₋₆—SO₃H, or —(CH₂)₁₋₆—O—PO₃H₂.

98. Insulin derivative according to any of paragraphs 3 or 78-97,wherein the insulin derivative are selected from the group consistingof:

-   N^(εB29)-10-(4-carboxyphenoxy)-decanoyl-γ-L-glutamylamide desB30    human insulin,-   N^(εB29)-4-[11-(4-Carboxyphenyl)undecanoylamino]butyryl desB30 human    insulin and-   N^(εB29)-{4-[10-(4-Carboxy-phenoxy)-decanoylamino]-butyryl} desB30    insulin

99. Insulin derivative according to paragraphs 1 or 2, wherein Q₁ is anamino acid amide of an amino acid with a carboxylic acid in the sidechain, or an amino acid with an uncharged side chain, the amino acidamide residue or amino acid residue having from 2 to 10 carbon atoms.

100. Insulin derivative according to any of paragraphs 1, 2 or 99,wherein Q₁ is selected from the group consisting of β-D-Asp-amide,β-L-Asp-amide, γ-L-Glu-amide and γ-D-Glu-amide.

101. Insulin derivative according to paragraph 1 or 2, wherein Q₁ is achain composed of two, three or four α-amino acid amide or amino acidresidues with uncharged side chains.

102. Insulin derivative according to paragraphs 1, 2 or 101, wherein Q₁is a chain of two amino acid amide residues selected from the groupconsisting of β-L-Asp-amide-β-L-Asp-amide, β-L-Asp-amide-γ-L-Glu-amide,γ-L-Glu-amide-γ-L-Glu-amide, γ-L-Glu-amide-β-L-Asp-amide,β-L-Asp-amide-β-D-Asp-amide, β-L-Asp-amide-γ-D-Glu-amide,γ-L-Glu-amide-γ-D-Glu-amide, γ-L-Glu-amide-β-D-Asp-amide,β-D-Asp-amide-β-L-Asp-amide, β-D-Asp-amide-γ-L-Glu-amide,γ-D-Glu-amide-γ-L-Glu-amide, γ-D-Glu-amide-β-L-Asp-amide,β-D-Asp-amide-β-D-Asp-amide, β-D-Asp-amide-γ-D-Glu-amide,γ-D-Glu-amide-γ-D-Glu-amide, γ-D-Glu-amide-β-D-Asp-amide.

103. Insulin derivative according to any of paragraphs 99-102, whereinQ₂ is a bond

104. Insulin derivative according to any of paragraphs 1, 2 or 99-102,wherein Q₂ is selected from the group consisting of—CO—((CR⁵R⁶)₁₋₆—NH—CO)— and —(CO—(CR⁵R⁶)₁₋₆—CO—NH)₁₋₄—, where R⁵independently can be H, —CH₃, —(CH₂)₁₋₆CH₃ or —CONH₂ and R⁶independently can be H, —CH₃, —(CH₂)₁₋₆CH₃;

105. Insulin derivative according to any of paragraphs 1, 2 or 99-104,wherein Q₂ is —CO—CH₂—(CHCONH₂)—NH—CO; —CO—(CH₂)₂—(CHCONH₂)—NH—CO)— or—CO—(CH₂)₃—(CHCONH₂)—NH—CO)—

106. Insulin derivative according to paragraphs 1, 2 or 104-105, whereinQ₁ is a bond.

107. Insulin derivative according any of paragraphs 99-106, wherein oneof Q₃, Q₄, or Q₅ is —(CH₂)_(m)— where m is an integer in the range of 1to 32 or 1-12.

108. Insulin derivative according to paragraph 107, wherein m is 4, 5,6, 8, 9, 10 or 11.

109. Insulin derivative according to any of paragraphs 99-108, whereinone of Q₃, Q₄, or Q₅ is (CH₂CH₂O)_(y)—; (CH₂CH₂CH₂O)_(y)—;(CH₂CH₂CH₂CH₂O)_(y)—; (CH₂CH₂OCH₂CH₂CH₂CH₂O)_(y)— or(CH₂CH₂CH₂OCH₂CH₂CH₂CH₂O)_(y)—; —(CH₂OCH₂)_(y)— where y is 1-20.

110. Insulin derivative according to paragraph 109, wherein one of Q₃,Q₄, or Q₅ is (CH₂CH₂O)_(y)— or (CH₂CH₂OCH₂CH₂CH₂CH₂O)_(y) wherein y isin the range of 1-12, 2-4 or 2-3

111. Insulin derivative according to any of paragraphs 109-110, whereiny is 1.

112. Insulin derivative according to any of paragraphs 99-111, whereinX₁, X₂ and

X₃ are independently of each other

O;

—C═O

a bond;

NCOR¹, where R¹ can be H, —CH₃ or —(CH)₁₋₆—CH₃; or

where R is hydrogen, C₁₋₃-alkyl, C₂₋₃-alkenyl or C₂₋₃-alkynyl;

113. Insulin derivative according to any of paragraphs 99-112, whereinX₁, X₂ and X₃ are:

where R is hydrogen, C₁₋₃-alkyl, C₂₋₃-alkenyl or C₂₋₃-alkynyl;

114. Insulin derivative according to any of paragraphs 99-113, whereinX₁ is

where R is H and wherein X₂ is

where R is H.

115. Insulin derivative according to any of paragraphs 99-114, wherein nis 0, 1, 2 or 3.

116. Insulin derivative according to any of paragraphs 99-115, wherein Zis —COOH.

117. Insulin derivative according to any of paragraphs 99-115, wherein Zis —CH(COOH)₂.

118. An insulin derivative according to any of paragraphs 99-115,wherein Z is —N(CH₂COOH)₂.

119. An insulin derivative according to any of paragraphs 99-115,wherein Z is —SO₃H.

120. An insulin derivative according to any of paragraphs 99-115,wherein Z is —PO₃H.

121. Insulin derivative according to any of paragraphs 1-2 or 99-120wherein the insulin derivative is

-   N^(εB29)-(3-(3-{4-[3-(7-carboxyheptanoylamino)propoxy]butoxy}propylcarbamoyl)-propionyl-γ-glutamylamide)    desB30 human Insulin.

In one aspect of the invention Q₁ is an amino acid amide of an aminoacid with a carboxylic acid in the side chain, or an amino acid with anuncharged side chain, the amino acid amide residue or amino acid residuehaving from 2 to 10 carbon atoms.

Q₁ can also be a chain composed of two, three or four amino acid amideof an amino acid with a carboxylic acid in the side chain and/or anamino acid with an uncharged side chain The chain of amino acids maycomprise at least one amino acid containing an amide. Thus, Q₁ can, forexample, be selected from the group comprising β-L-Asp-amide,β-D-Asp-amide, γ-L-Glu-amide and γ-D-Glu-amide.

In one aspect Q₁ is a chain composed of two uncharged amino acidresidues, which has from 4 to 10 carbon atoms. The amino acids cancomprise an amide. Examples of such amino acid residues areβ-L-Asp-amide-β-L-Asp-amide, β-L-Asp-amide-γ-L-Glu-amide,γ-L-Glu-amide-γ-L-Glu-amide, γ-L-Glu-amide-β-L-Asp-amide,β-L-Asp-amide-β-D-Asp-amide, β-L-Asp-amide-γ-D-Glu-amide,γ-L-Glu-amide-γ-D-Glu-amide, γ-L-Glu-amide-β-D-Asp-amide,β-D-Asp-amide-β-L-Asp-amide, β-D-Asp-amide-γ-L-Glu-amide,γ-D-Glu-amide-γ-L-Glu-amide, γ-D-Glu-amide-β-L-Asp-amide,β-D-Asp-amide-β-D-Asp-amide, β-D-Asp-amide-γ-D-Glu-amide,γ-D-Glu-amide-γ-D-Glu-amide, γ-D-Glu-amide-β-D-Asp-amide,

In one aspect of the invention Q₁ is a chain composed of three aminoacid residues, independently having from 4 to 10 carbon atoms, where atleast one of the amino acid residues of the chain being selected fromthe group of residues having an amide. The combination of the threeamino acid amides can be any combination of β-L-Asp-amide,β-D-Asp-amide, γ-L-Glu-amide and γ-D-Glu-amide, which means that 64different combinations are possible.

In a further aspect, Q₁ is a chain composed of four amino acid residues,independently having from 4 to 10 carbon atoms, where at least one ofthe amino acid residues of the chain being selected from the group ofresidues having an amide. The combination of the four amino acid amidescan be any combination of β-L-Asp-amide, β-D-Asp-amide, γ-L-Glu-amideand γ-D-Glu-amide, which means that 256 different combinations arepossible.

122. An insulin derivative having a substituent attached to desB30 humaninsulin at either the α-amino group of the N-terminal amino acid residueof the B chain or at an ε-amino group of a Lys residue present in the Aor B chain of desB30 human insulin, which substituent comprises at leastone fatty difunctionalized moiety with about 6 to about 32 carbon atomsand an uncharged linker of the formula —CO—((CH₂)₁₋₆—NH—CO)₁₋₄ whichlinks the fatty difunctionalized moiety to desB30 human insulin, whereinthe fatty difunctionalized moiety comprises an aromatic group.

123. Insulin derivative according to paragraph 122, wherein theuncharged linker is selected from the group consisting of:—CO—CH₂—NH—CO, —CO—(CH₂)₂—NH—CO, —CO—(CH₂)₃—NH—CO and —CO—(CH₂)₁—NH—CO.

124. Insulin derivative according to paragraphs 122 or 123, wherein thefatty difunctionalized moiety is

T-(CH₂)_(s)—Y₁—(Ar)_(v1)—Y₂—(CH₂)_(w)—Y₃—(Ar)_(v2)—Y₄—(CH₂)_(t)—Y₅—(Ar)_(v3)—Y₆—(CH₂)_(z)-Twherein

Ar can be arylene or heteroarylene, which may be substituted with one ortwo groups selected from the group consisting of —CH₃, —(CH)₁₋₆—CH₃,—CONR¹R² or —SO₂NR¹R² where R¹ and R², independently of each other canbe H, —CH₃ or —(CH)₁₋₆—CH₃;

Y₁-Y₆ independently of each other can be O, S, S═O, SO₂ or a bond;

s, w, t and z independently of each other are zero or an integer from 1to 10 so that the sum of s, w, t and z is in the range from 4 to 30,

v₁, v₂, and v₃ independently of each other can be zero or 1 with theproviso that Y₁-Y₆ do not link to each other and that the structure—O—(CH₂)₁—O— does not occur; and

T is a functional group selected from carboxy, amino or hydroxyl groups.

125. Insulin derivative according to paragraphs 122-124, wherein theinsulin derivative isN^(εB29)-4-[11-(4-Carboxyphenyl)undecanoylamino]butyryl desB30 humaninsulin.

126. An insulin derivative having a substituent attached to a parentinsulin moiety at either the α-amino group of the N-terminal amino acidresidue of the B chain or at an ε-amino group of a Lys residue presentin the A or B chain of the parent insulin moiety, which substituentcomprises at least one fatty difunctionalized moiety with about 6 toabout 32 carbon atoms and an uncharged linker which links the fattydifunctionalized moiety to the parent insulin, provided that theuncharged linker does not comprise —(CO—(CH₂)₁₋₆—NH—CO)₁₋₄— if theinsulin substituent comprises an aromatic group and that the substituentdoes not comprise one or more residues selected from the group of—CO—(CH₂)₂—CO—NH—(CH₂CH₂O)₂—, —CO—(CH₂)₂—CO—NH—CH₂—(CH₂CH₂O)₂—,—CO—(CH₂)₂—CO—NH—(CH₂CH₂O)₃— and—CO—(CH₂)₂—CO—NH—CH₂—(CH₂CH₂OCH₂CH₂CH₂CH₂O)—.

127. An insulin derivative having a substituent attached to a parentinsulin moiety at either the α-amino group of the N-terminal amino acidresidue of the B chain or at an ε-amino group of a Lys residue presentin the A or B chain of the parent insulin moiety, which substituentcomprises at least one fatty difunctionalized moiety with about 6 toabout 32 carbon atoms and an uncharged linker which links the fattydifunctionalized moiety to the parent insulin, provided that thesubstituent does not comprise one or more residues selected from thegroup of —CO—(CH₂)₂—CO—NH—(CH₂CH₂O)₂—, —CO—(CH₂)₂—CO—NH—CH₂—(CH₂CH₂O)₂—,—CO—(CH₂)₂—CO—NH—(CH₂CH₂O)₃— and—CO—(CH₂)₂—CO—NH—CH₂—(CH₂CH₂OCH₂CH₂CH₂CH₂O)—.

128. An insulin derivative having a substituent attached to a parentinsulin moiety at either the α-amino group of the N-terminal amino acidresidue of the B chain or at an ε-amino group of a Lys residue presentin the A or B chain of the parent insulin moiety, which substituentcomprises at least one fatty difunctionalized moiety with about 6 toabout 32 carbon atoms and an uncharged linker which links the fattydifunctionalized moiety to the parent insulin, provided that thesubstituent does not comprise one or more residues selected from thegroup of —CO—(CH₂)₂—CO—NH—(CH₂CH₂O)₂—, —CO—(CH₂)₂—CO—NH—CH₂—(CH₂CH₂O)₂—,—CO—(CH₂)₂—CO—NH—(CH₂CH₂O)₃— and—CO—(CH₂)₂—CO—NH—CH₂—(CH₂CH₂OCH₂CH₂CH₂CH₂O)— characterized by theuncharged linker being selected from the group consisting of

-   -   an amino acid amide of an amino acid with a carboxylic acid in        the side chain, or an amino acid with an uncharged side chain,        which residue forms, with its carboxylic acid group, an amide        group together with the α-amino group of the N-terminal amino        acid residue of the B chain of Ins or together with the ε-amino        group of a Lys residue present in the A or B chain of Ins, or

a chain composed of two, three or four α-amino acid amide or amino acidresidues as specified above linked together via amide bonds, whichchain—via an amide bond—is linked to the α-amino group of the N-terminalamino acid residue of the B chain of Ins or to the ε-amino group of aLys residue present in the A or B chain of Ins

—COCH(CONH₂)—

—COCH₂N(CH₂CONH₂)—

—COCH₂N(CH₂CONH₂)COCH₂N(CH₂CONH₂)

—COCH₂CH₂N(CH₂CH₂CONH₂)—

—COCH₂CH₂N(CH₂CH₂CONH₂)—COCH₂CH₂N(CH₂CH₂CONH₂)—

—COCH₂N(CH₂CH₂CONH₂)—

—COCH₂CH₂N(CH₂CONH₂)—

—COCH₂OCH₂CONH—; or

—CO—((CR⁵R⁶)₁₋₆—CO—NH)₁₋₄—, where R⁵ independently can be H, —CH₃,—(CH₂)₁₋₆CH₃ or —CONH₂ and R⁶ independently can be H, —CH₃, —(CH₂)₁₋₆CH₃

provided that if an amine in the uncharged linker forms a bond with therest of the substituent, the amine must be bound to the rest of thesubstituent via a carbonyl group.

129. An insulin derivative having a substituent attached to a parentinsulin moiety at either the α-amino group of the N-terminal amino acidresidue of the B chain or at an ε-amino group of a Lys residue presentin the A or B chain of the parent insulin moiety, which substituentcomprises at least one fatty difunctionalized moiety with about 6 toabout 32 carbon atoms and an uncharged linker which links the fattydifunctionalized moiety to the parent insulin, provided that theuncharged linker does not comprise —(CO—(CH₂)₁₋₆—NH—CO)₁₋₄— if theinsulin substituent comprises an aromatic group and that the substituentdoes not comprise one or more residues selected from the group of—CO—(CH₂)₂—CO—NH—(CH₂CH₂O)₂—, —CO—(CH₂)₂—CO—NH—CH₂—(CH₂CH₂O)₂—,—CO—(CH₂)₂—CO—NH—(CH₂CH₂O)₃— and—CO—(CH₂)₂—CO—NH—CH₂—(CH₂CH₂OCH₂CH₂CH₂CH₂O)— characterized by theuncharged linker being selected from the group consisting of

-   -   an amino acid amide of an amino acid with a carboxylic acid in        the side chain, or an amino acid with an uncharged side chain,        which residue forms, with its carboxylic acid group, an amide        group together with the α-amino group of the N-terminal amino        acid residue of the B chain of Ins or together with the ε-amino        group of a Lys residue present in the A or B chain of Ins, or

a chain composed of two, three or four α-amino acid amide or amino acidresidues as specified above linked together via amide bonds, whichchain—via an amide bond—is linked to the α-amino group of the N-terminalamino acid residue of the B chain of Ins or to the ε-amino group of aLys residue present in the A or B chain of Ins,

—COCH(CONH₂)—

—COCH₂N(CH₂CONH₂)—

—COCH₂N(CH₂CONH₂)COCH₂N(CH₂CONH₂)

—COCH₂CH₂N(CH₂CH₂CONH₂)—

—COCH₂CH₂N(CH₂CH₂CONH₂)—COCH₂CH₂N(CH₂CH₂CONH₂)—

—COCH₂N(CH₂CH₂CONH₂)—

—COCH₂CH₂N(CH₂CONH₂)—

—COCH₂OCH₂CONH—

—CO—((CR⁵R⁶)₁₋₆—NH—CO)₁₋₄—; or

—CO—((CR⁵R⁶)₁₋₆—CO—NH)₁₋₄—, where R⁵ independently can be H, —CH₃,—(CH₂)₁₋₆CH₃ or —CONH₂ and R⁶ independently can be H, —CH₃,—(CH₂)₁₋₆CH₃,

provided that if an amine in the uncharged linker forms a bond with therest of the substituent, the amine must be bound to the rest of thesubstituent via a carbonyl group.

130. An insulin derivative having a substituent attached to a parentinsulin moiety at either the α-amino group of the N-terminal amino acidresidue of the B chain or at an ε-amino group of a Lys residue presentin the A or B chain of the parent insulin moiety, which substituentcomprises at least one fatty difunctionalized moiety with about 6 toabout 32 carbon atoms and an uncharged linker which links the fattydifunctionalized moiety to the parent insulin, provided that thesubstituent does not comprise one or more residues selected from thegroup of —CO—(CH₂)₂—CO—NH—(CH₂CH₂O)₂—, —CO—(CH₂)₂—CO—NH—CH₂—(CH₂CH₂O)₂—,—CO—(CH₂)₂—CO—NH—(CH₂CH₂O)₃— and—CO—(CH₂)₂—CO—NH—CH₂—(CH₂CH₂OCH₂CH₂CH₂CH₂O)— characterized by theuncharged linker being selected from the group consisting of

-   -   an amino acid amide of an amino acid with a carboxylic acid in        the side chain, or an amino acid with an uncharged side chain,        which residue forms, with its carboxylic acid group, an amide        group together with the α-amino group of the N-terminal amino        acid residue of the B chain of Ins or together with the ε-amino        group of a Lys residue present in the A or B chain of Ins, or

a chain composed of two, three or four α-amino acid amide or amino acidresidues as specified above linked together via amide bonds, whichchain—via an amide bond—is linked to the α-amino group of the N-terminalamino acid residue of the B chain of Ins or to the ε-amino group of aLys residue present in the A or B chain of Ins,

—COCH(CONH₂)—

—COCH₂N(CH₂CONH₂)—

—COCH₂N(CH₂CONH₂)COCH₂N(CH₂CONH₂)

—COCH₂CH₂N(CH₂CH₂CONH₂)—

—COCH₂CH₂N(CH₂CH₂CONH₂)—COCH₂CH₂N(CH₂CH₂CONH₂)—

—COCH₂N(CH₂CH₂CONH₂)—

—COCH₂CH₂N(CH₂CONH₂)—

—COCH₂OCH₂CONH—

—CO—((CR⁵R⁶)₁₋₆—NH—CO)₁₋₄—; or

—CO—((CR⁵R⁶)₁₋₆—CO—NH)₁₋₄—, where R⁵ independently can be H, —CH₃,—(CH₂)₁₋₆CH₃ or —CONH₂ and R⁶ independently can be H, —CH₃, —(CH₂)₁₋₆CH₃

provided that if an amine in the uncharged linker forms a bond with therest of the substituent, the amine must be bound to the rest of thesubstituent via a carbonyl group.

131. An insulin derivative having a substituent attached to a parentinsulin moiety at either the α-amino group of the N-terminal amino acidresidue of the B chain or at an ε-amino group of a Lys residue presentin the A or B chain of the parent insulin moiety, which substituentcomprises at least one fatty difunctionalized moiety with about 6 toabout 32 carbon atoms and an uncharged linker which links the fattydifunctionalized moiety to the parent insulin, provided that anon-linking amide is present in the linker if the substituent comprisesan aromatic group and that the substituent does not comprise one or moreresidues selected from the group of —CO—(CH₂)₂—CO—NH—(CH₂CH₂O)₂—,—CO—(CH₂)₂—CO—NH—CH₂—(CH₂CH₂O)₂—, —CO—(CH₂)₂—CO—NH—(CH₂CH₂O)₃— and—CO—(CH₂)₂—CO—NH—CH₂—(CH₂CH₂OCH₂CH₂CH₂CH₂O)—.

132. An insulin derivative having a substituent attached to a parentinsulin moiety at either the α-amino group of the N-terminal amino acidresidue of the B chain or at an ε-amino group of a Lys residue presentin the A or B chain of the parent insulin moiety, which substituentcomprises at least one fatty difunctionalized moiety with about 6 toabout 32 carbon atoms and an uncharged linker which links the fattydifunctionalized moiety to the parent insulin, provided that the fattydifunctionalized moiety is an aliphatic chain, an non-linking amide ispresent in the linker.

133. Insulin derivative according to paragraph 121-129, wherein thesubstituent is attached to the α-amino group of the N-terminal aminoacid residue of the B chain of the parent insulin.

134. Insulin derivative according to paragraph 121-129, wherein thesubstituent is attached to the ε-amino group of a Lys residue present inthe A or B chain of the parent insulin.

135. Insulin derivative according to paragraphs 121-129 or 131 whereinthe substituent is attached to the ε-amino group of the Lys residue inposition B29 present in the B chain of the parent insulin.

136. Insulin derivative according to paragraph 132, wherein thesubstituent is attached to the ε-amino group of the Lys residue inposition B29 in desB30 human insulin

137. Insulin derivative according to any of paragraphs 121-129, whereinthe linker comprises an amide or an N-substituted amide of the formula—CONR⁷R⁸ where R⁷ and R⁸, independently of each other can be hydrogen,methyl, ethyl, propyl or isopropyl.

138. Insulin derivative according to paragraph 134, where R⁷ and R⁸ arehydrogen.

139. Insulin derivative according to paragraph 121-129, wherein thelinker comprises a non-linking amide.

140. Insulin derivative according to any of paragraphs 121-129, whereinthe substituent comprises one or more residues of ethyleneglycol,propyleneglycol and/or butyleneglycol containing independently at eachtermini a group selected from —NH₂ and —COOH, which is used to connectthe individual components of the substituent.

141. Insulin derivative according to paragraphs 121-129, wherein thesubstituent comprises at least one aromatic group.

142. Insulin derivative according to any of the paragraphs 1-141,wherein the parent insulin is human insulin or porcine insulin.

143 Insulin derivative according to any of the paragraphs 1-141, whereinthe parent insulin is an insulin analogue.

144. Insulin derivative according to any of paragraphs 143, wherein theamino acid residue at position B30 of the parent insulin is Lys or hasbeen deleted.

145. Insulin derivative according to paragraphs 143-44, wherein theparent insulin is desB30 human insulin.

146. Insulin derivative according to any of paragraphs 143-145, whereinthe amino acid residue at position B1 of the parent insulin has beendeleted.

147. Insulin derivative according to any of paragraphs 143-146, whereinthe amino acid residue in position A21 of the parent insulin is Gly orAsn.

148. Insulin derivative according to any of paragraphs 143-147, whereinthe amino acid residue at position B3 of the parent insulin is Lys.

149. Insulin derivative according to any of paragraphs 143-148, whereinthe amino acid residue at position B28 of the parent insulin is Asp orLys.

150. Insulin derivative according to any of paragraphs 143-149, whereinthe amino acid residue at position B29 of the parent insulin is Pro orThr.

151. Insulin derivative according to paragraph 149, wherein the parentinsulin is AspB28 human insulin.

152. Insulin derivative according to paragraph 147, wherein the parentinsulin is GlyA21 human insulin or GlyA21desB30 human insulin orGlyA21ArgB31ArgB32 human insulin.

153. Insulin derivative according to paragraph 148, wherein the parentinsulin is LysB3GluB29 human insulin.

154. Insulin derivative according to paragraph 149-150, wherein theparent insulin is LysB28ProB29 human insulin.

155. Insulin derivative according to paragraph 144 and 150, wherein theparent insulin is ThrB29LysB30 human insulin.

156. A zinc complex of an insulin derivative according to any one of thepreceding paragraphs wherein two zinc ions, three zinc ions, four zincions, five zinc ions, six zinc ions, seven zinc ions, eight zinc ions,nine zinc ions or ten six zinc ions are bound per six molecules ofinsulin derivative.

157. A pharmaceutical composition for the treatment of diabetes in apatient in need of such treatment, comprising a therapeuticallyeffective amount of an insulin derivative according to paragraphs 1-155or a zinc complex according to paragraph 156 together with apharmaceutically acceptable carrier.

158. A pharmaceutical composition for the treatment of diabetes in apatient in need of such treatment, comprising a therapeuticallyeffective amount of an insulin derivative according to paragraphs 1-155or a zinc complex according to paragraph 156 in mixture with an insulinor an insulin analogue which has a rapid onset of action, together witha pharmaceutically acceptable carrier.

159. A method of treating diabetes in a patient in need of such atreatment, comprising administering to the patient a therapeuticallyeffective amount of an insulin derivative according to paragraphs 1-155or a zinc complex according to paragraph 156 together with apharmaceutically acceptable carrier.

160. A method of treating diabetes in a patient in need of such atreatment, comprising administering to the patient a therapeuticallyeffective amount of an insulin derivative according to paragraphs 1-155or a zinc complex according to paragraph 156 in mixture with an insulinor an insulin analogue which has a rapid onset of action, together witha pharmaceutically acceptable carrier.

161. A method according to paragraphs 138 or 139 for pulmonary treatmentof diabetes.

162. Use of an insulin derivative according to paragraphs 1-155 or azinc complex according to paragraph 156 for the manufacture of apharmaceutical composition for the use in the treatment of type 1diabetes, type 2 diabetes and other states that cause hyperglycaemia.

163. Use of an insulin derivative according to paragraphs 1-155 or azinc complex according to paragraph 156 in mixture with an insulin or aninsulin analogue which has a rapid onset of action for the manufactureof a pharmaceutical composition for the use in the treatment of type 1diabetes, type 2 diabetes and other states that cause hyperglycaemia.

164. A mixture of an insulin derivative according to paragraphs 1-155 ora zinc complex according to paragraph 156 and a rapid acting insulinanalogue selected group consisting of AspB28 human insulin; LysB28ProB29human insulin and LysB3GluB29 human insulin.

165. An insulin derivative, wherein the insulin derivative is selectedfrom the group consisting of:

-   N^(εB29)-ω-carboxy-pentadecanoyl-γ-L-glutamylamide desB30 human    insulin,-   N^(εB29)-ω-carboxy-pentadecanoyl-γ-amino-butanoyl desB30 human    insulin,-   N^(εB29)-ω-carboxy-tetradecanoyl-γ-L-glutamylamide desB30 human    insulin,-   N^(εB29)-ω-carboxy-tridecanoyl-γ-L-glutamylamide desB30 human    insulin,-   N^(εB29)-ω-carboxy-pentadecanoyl-β-alanyl desB30 human insulin,-   N^(εB29)-ω-carboxy-pentadecanoyl-γ-L-aspartylamide desB30 human    insulin,-   N^(εB29)-ω-carboxy-pentadecanoyl-ε-aminohexanoyl desB30 human    insulin,-   N^(εB29)-ω-carboxy-pentadecanoyl-δ-aminopentanoyl desB30 human    insulin,-   N^(εB29)-10-(4-carboxyphenoxy)-decanoyl-γ-L-glutamylamide desB30    human insulin,-   N^(εB29)-4-[11-(4-Carboxyphenyl)undecanoylamino]butyryl desB30 human    insulin,-   N^(εB29)-(3-(3-{4-[3-(7-carboxyheptanoylamino)propoxy]butoxy}propylcarbamoyl)-propionyl-γ-glutamylamide)    desB30 human Insulin,-   N^(εB29)-ω-carboxy-tridecanoyl-γ-amino-butanoyl desB30 human    insulin,-   N^(εB29)-ω-carboxy-undecanoyl-γ-amino-butanoyl desB30 human insulin,-   N^(εB29)-ω-carboxy-tetradecanoyl-γ-amino-butanoyl desB30 human    insulin,-   N^(εB29)-{4-[10-(4-Carboxy-phenoxy)-decanoylamino]-butyryl} desB30    insulin,-   N^(εB29)-{4-[(14-Carboxy-tetradecanoylamino)-methyl]-benzoyl} desB30    insulin,-   N^(εB29)-[16-(4-Carboxy-phenoxy)-hexadecanoyl] desB30 insulin,-   N^(εB29)-{4-[(15-carboxypentadecanoylamino)benzoyl]-desB30 human    insulin and-   N^(εB29)-{4-[(15-Carboxy-pentadecanoylamino)-methyl]-benzoyl}-desB30    insulin

166. Insulin derivative as described in the examples.

The invention will further be summarized in the following paragraphs:

1a. Insulin derivative having a substituent attached to a parent insulinmoiety at either the α-amino group of the N-terminal amino acid residueof the B chain or at an ε-amino group of a Lys residue present in the Bchain of the parent insulin moiety, which substituent comprises at leastone fatty difunctionalized moiety with about 6 to about 32 carbon atomsand an uncharged linker which link the fatty difunctionalized moiety tothe parent insulin, provided that the uncharged linker does not comprise—(CO—(CH₂)₂₋₆—NH—CO)₁₋₄— if the insulin substituent comprises anaromatic group.

2a. Insulin derivative according to paragraphs 1a, wherein thesubstituent is attached to the α-amino group of the N-terminal aminoacid residue of the B-chain of the parent insulin.

3a. Insulin derivative according to paragraphs 1a, wherein thesubstituent is attached to the ε-amino group of a Lys residue present inthe B-chain of the parent insulin.

4a. Insulin derivative according to paragraphs 1a or 3a wherein thesubstituent is attached to the ε-amino group of the Lys residue inposition B29 present in the B-chain of the parent insulin.

5a. Insulin derivative according to paragraphs 4a, wherein thesubstituent is attached to the ε-amino group of the Lys residue inposition B29 in LysB29desB30 human insulin

6a. Insulin derivative according to any of paragraphs 1a-5a, wherein thelinker comprises an amide or a N-substituted amide of the formula—CONR₇R₈, where R₇ and R₈, independently of each other can be hydrogen,methyl, ethyl, propyl or isopropyl.

7a. Insulin derivative according to paragraphs 6a, where R₇ and R₈ arehydrogen.

8a. Insulin derivative according to paragraphs 1, wherein the linkercomprises an amide.

9a. Insulin derivative according to any of paragraphs 1a-7a, wherein thesubstituent comprises one or more residues of ethyleneglycol,propyleneglycol and/or butyleneglycol containing independently at eachtermini a group selected from —NH₂ and —COOH.

10a. Insulin derivative according to paragraphs 1a-7a, wherein thesubstituent comprises at least one aromatic group.

11a. Insulin derivatives according to paragraphs 1a having the formula

wherein Ins is the parent insulin moiety which via the α-amino group ofthe N-terminal amino acid residue of the B chain or an ε-amino group ofa Lys residue present in the B chain of the insulin moiety is bound tois bound to Q₁ or Q₂ in the substituent;each n is independently 0, 1, 2, 3, 4, 5 or 6;

Q₁ is:

-   -   an α-amino acid amide residue having a carboxylic acid group in        the substituent which residue forms, with one of its carboxylic        acid groups, an amide group together with the α-amino group of        the N-terminal amino acid residue of the B chain or together        with the ε-amino group of a Lys residue present in the B chain        of the parent insulin;    -   a chain composed of two, three or four α-amino acid amide        residues linked together via amide bonds, which chain—via an        amide bond—is linked to the α-amino group of the N-terminal        amino acid residue of the B chain or to the ε-amino group of a        Lys residue present in the B chain of the parent insulin, the        amino acid residues of W being selected from the group of amino        acid residues having a neutral substituent and amino acid        residues having a carboxylic acid group in the substituent so        that W has at least one amino acid residue which has a        carboxylic acid group in the substituent; or    -   a bond

Q₂ is:

—COCH(CONH₂)—

—COCH₂N(CH₂CONH₂)—

—COCH₂N(CH₂CONH₂)COCH₂N(CH₂CONH₂)

—COCH₂CH₂N(CH₂CH₂CONH₂)—

—COCH₂CH₂N(CH₂CH₂CONH₂)—COCH₂CH₂N(CH₂CH₂CONH₂)—

—COCH₂N(CH₂CH₂CONH₂)—

—COCH₂CH₂N(CH₂CONH₂)—

—(CO—(CH₂)₂₋₆—NH—CO)₁₋₄—;

—(CO—(CH₂)₂₋₆—CO—NH)₁₋₄—;

—(CO—(CR₉R₁₀)₁₋₆—CO—NH)₁₋₄—, where R₉ and R₁₀, independently of eachother can be H, —CH₃, —(CH₂)₁₋₆CH₃ or —CONH₂; or

a bond

-   -   provided that at least one of Q₁ or Q₂ is not a bond;        Q₃, Q₄, and Q₅ independently of each other can be    -   —(CH₂)_(m)— where m is an integer in the range of 1 to 32;    -   a divalent hydrocarbon chain comprising 1, 2 or 3 —CH═CH— groups        and a number of —CH₂— groups sufficient to give a total number        of carbon atoms in the chain in the range of 4 to 32;    -   (CH₂CH₂O)_(y)—; (CH₂CH₂CH₂O)_(y)—; (CH₂CH₂CH₂CH₂O)_(y)—;        (CH₂CH₂OCH₂CH₂CH₂CH₂O)_(y)— or (CH₂CH₂CH₂OCH₂CH₂CH₂CH₂O)_(y)—;        —(CH₂OCH₂)_(y)— where y is 1-20;    -   —(CR₃R₄)₁₋₆—(NHCO—(CR₃R₄)₁₋₆—NHCO)₁₋₂—(CR₃R₄)₁₋₆ or        —(CR₃R₄)₁₋₆—(CONH—(CR₃R₄)₁₋₆—CONH)₁₋₂—(CR₃R₄)₁₋₆—,        —(CR₃R₄)₁₋₆—(NHCO—(CR₃R₄)₁₋₆—CONH)₁₋₂—(CR₃R₄)₁₋₆— or        —(CR₃R₄)₁₋₆—(CONH—(CR₃R₄)₁₋₆—NHCO)₁₋₂—(CR₃R₄)₁₋₆ where R₃ and R₄        independently of each other and independently for each carbon        can be H, —COOH or OH,    -   —(CR₅R₆)₁₋₆—, where R₅ and R₆ independently of each other and        independently for each carbon can be H, —COOH, (CH₂)₁₋₆COOH;    -   —((CR₁R₂)₁₋₆—NR₁₅—CO)₁₋₄—, where R₁, R₂ and R₁₅ independently of        each other can be H, —CH₃, —CH₁₋₆CH₃, —SO₃H, —(CH₂)₁₋₆—SO₃H,        —(CH₂)₁₋₆—O—PO₃H₂ or CONH₂ and R₁₅ can be arylene which may be        substituted with one or two groups of R₁, R₂ as defined above;    -   NR₁₅ where R₁₅ is defined as above;    -   arylene or heteroarylene, which may be substituted with one or        two groups selected from the group consisting of —COOH, —CH₃,        —CH₁₋₆CH₃, —SO₃H, —(CH₂)_(p)—SO₃H, —CONR₁R₂ or —SO₂NR₁R₂, where        R₁ and R₂, independently of each other can be H, —CH₃,        —(CH)₁₋₆—CH₃, —SO₃H, —(CH₂)₁₋₆—SO₃H, —(CH₂)₁₋₆—O—PO₃H₂ or CONH₂;    -   a divalent hydrocarbon chain of the formula

—(CH₂)_(s)—Y₁—(C₆H₄)_(v1)—Y₂—(CH₂)_(w)—Y₃—(C₆H₄)_(v2)—Y₄—(CH₂)_(t)—Y₅—(C₆H₄)_(v3)—Y₆—(CH₂)_(z)—

wherein Y₁-Y₆ independently of each other can be O; S or a bond; wheres, w, t and z independently of each other are zero or an integer from 1to 10 so that the sum of s, w, t and z is in the range from 4 to 30, andv₁, v₂, and v₃ independently of each other can be zero or 1 with theproviso that Y₁-Y₆ do not link to each other; or

-   -   a bond;        -   with the proviso that Q₃-Q₅ are different;            X₁, X₂ and X₃ are independently of each other:

O;

—C═O

a bond; or

-   -   where R is hydrogen, C₁₋₃-alkyl, C₂₋₃-alkenyl or C₂₋₃-alkynyl;        and

Z is:

—COOH;

—CO-Asp;

—CO-Glu;

—CO-Gly;

—CO-Sar;

—CH(COOH)₂;

—N(CH₂COOH)₂;

—SO₃H

—OSO₃H

—OPO3H₂

—PO₃H₂ or

-tetrazol-5-yl or

—O—W₁,

-   -   where W₁ is arylene or heteroarylene, which may be substituted        with one or two groups selected from the group consisting of        —COOH, —SO₃H, —(CH₂)₁₋₆—SO₃H, —CONR₁₃R₁₄ or —SO₂NR₁₂R₁₄, where        R₁₃ and R₁₄, independently of each other can be H, —SO₃H,        —(CH₂)₁₋₆—SO₃H, —(CH₂)₁₋₆—O—PO₃H₂, —CONH₂ or tetrazo-5-lyl; and        any Zn²⁺ complex thereof.

12a. Insulin derivative according to paragraphs 11a, wherein Q₁ is anamino acid amide residue having from 4 to 10 carbon atoms.

13a. Insulin derivative according to any of paragraphs 11a-12a, whereinQ₁ is selected from the group consisting of β-D-Asp-amide,β-L-Asp-amide, γ-L-Glu-amide and γ-D-Glu-amide.

14a. Insulin derivative according to paragraphs 11a, wherein Q₁ is achain of amino acid amide residues.

15a. Insulin derivative according to paragraphs 11a or 14a, wherein Q₁is a chain of two amino acid amide residues selected from the groupconsisting of β-L-Asp-amide-β-L-Asp-amide, β-L-Asp-amide-γ-L-Glu-amide,γ-L-Glu-amide-γ-L-Glu-amide, γ-L-Glu-amide-β-L-Asp-amide,β-L-Asp-amide-β-D-Asp-amide, β-L-Asp-amide-γ-D-Glu-amide,γ-L-Glu-amide-γ-D-Glu-amide, γ-L-Glu-amide-β-D-Asp-amide,β-D-Asp-amide-β-L-Asp-amide, β-D-Asp-amide-γ-L-Glu-amide,γ-D-Glu-amide-γ-L-Glu-amide, γ-D-Glu-amide-β-L-Asp-amide,β-D-Asp-amide-β-D-Asp-amide, β-D-Asp-amide-γ-D-Glu-amide,γ-D-Glu-amide-γ-D-Glu-amide, γ-D-Glu-amide-β-D-Asp-amide,

16a. Insulin derivative according to any of paragraphs 11a-15a, whereinQ₂ is a bond

17a. Insulin derivative according to any of paragraphs 11a-15a, whereinQ₂ is selected from the group consisting of —(CO—(CH₂)₂₋₆—NH—CO)₁₋₄—;—(CO—(CH₂)₂₋₆—CO—NH)₁₋₄—;

18a. Insulin derivative according to paragraphs 17a, wherein Q₂ isselected from the group consisting of —(CO—(CH₂)₂—NH—CO)₁— or—(CO—(CH₂)₃—NH—CO)₁₋₄.

19a. Insulin derivative according to paragraphs 17a-18a, wherein Q₁ is abond.

20a. Insulin derivative according to paragraphs 11a wherein

Q₁ is:

-   -   an α-amino acid amide residue having a carboxylic acid group in        the substituent which residue forms, with one of its carboxylic        acid groups, an amide group together with the α-amino group of        the N-terminal amino acid residue of the B chain or together        with the ε-amino group of a Lys residue present in the B chain        of the parent insulin;    -   a chain composed of two, three or four α-amino acid amide        residues linked together via amide bonds, which chain—via an        amide bond—is linked to the α-amino group of the N-terminal        amino acid residue of the B chain or to the ε-amino group of a        Lys residue present in the B chain of the parent insulin, the        amino acid residues of W being selected from the group of amino        acid residues having a neutral substituent and amino acid        residues having a carboxylic acid group in the substituent so        that W has at least one amino acid residue which has a        carboxylic acid group in the substituent; or    -   a bond

Q₂ is:

—COCH(CONH₂)—

—COCH₂N(CH₂CONH₂)—

—COCH₂N(CH₂CONH₂)COCH₂N(CH₂CONH₂)

—COCH₂CH₂N(CH₂CH₂CONH₂)—

—COCH₂CH₂N(CH₂CH₂CONH₂)—COCH₂CH₂N(CH₂CH₂CONH₂)—

—COCH₂N(CH₂CH₂CONH₂)—

—COCH₂CH₂N(CH₂CONH₂)—

—(CO—(CH₂)₂₋₆—NH—CO)₁₋₄—;

—(CO—(CH₂)₂₋₆—CO—NH)₁₋₄—;

—(CO—(CR₉R₁₀)₁₋₆—CO—NH)₁₋₄—, where R₉ and R₁₀, independently of eachother can be H, —CH₃, —(CH₂)₁₋₆CH₃ or —CONH₂; or

a bond

-   -   provided that at least one of Q₁ or Q₂ is not a bond;    -   Q₃ is    -   —(CH₂)_(m)— where m is an integer in the range of 6 to 32;    -   a divalent hydrocarbon chain comprising 1, 2 or 3 —CH═CH— groups        and a number of —CH₂— groups sufficient to give a total number        of carbon atoms in the chain in the range of 4 to 32; or    -   a divalent hydrocarbon chain of the formula        —(CH₂)_(s)C₆H₄(CH₂)_(w)— wherein v and w are integers or one of        them is zero so that the sum of s and w is in the range of 6 to        30;        -   X₁, can be —C═O or a bond;        -   Q₄, Q₅, X₂ and X₃ are bonds;            All values of n are zero; and

Z is:

—COOH;

—CO-Asp;

—CO-Glu;

—CO-Gly;

—CO-Sar;

—CH(COOH)₂;

—N(CH₂COOH)₂;

—SO₃H

—OSO₃H

—OPO3H₂

—PO₃H₂ or

-tetrazol-5-yl

and any Zn²⁺ complex thereof

21a. Insulin derivative according to paragraphs 20a, wherein Q₁ is anamino acid amide residue having from 4 to 10 carbon atoms.

22a. Insulin derivative according to any of paragraphs 20a-21a, whereinQ₁ is selected from the group consisting of β-D-Asp-amide,β-L-Asp-amide, γ-L-Glu-amide and γ-D-Glu-amide.

23a. Insulin derivative according to paragraphs 20a, wherein Q₁ is achain of amino acid amide residues.

24a. Insulin derivative according to paragraphs 20a or 23a, wherein Q₁is a chain of two amino acid amide residues selected from the groupconsisting of β-L-Asp-amide-β-L-Asp-amide, β-L-Asp-amide-γ-L-Glu-amide,γ-L-Glu-amide-γ-L-Glu-amide, γ-L-Glu-amide-β-L-Asp-amide,β-L-Asp-amide-β-D-Asp-amide, β-L-Asp-amide-γ-D-Glu-amide,γ-L-Glu-amide-γ-D-Glu-amide, γ-L-Glu-amide-β-D-Asp-amide,β-D-Asp-amide-β-L-Asp-amide, β-D-Asp-amide-γ-L-Glu-amide,γ-D-Glu-amide-γ-L-Glu-amide, γ-D-Glu-amide-β-L-Asp-amide,β-D-Asp-amide-β-D-Asp-amide, β-D-Asp-amide-γ-D-Glu-amide,γ-D-Glu-amide-γ-D-Glu-amide, γ-D-Glu-amide-β-D-Asp-amide,

25a. Insulin derivative according to any of paragraphs 20a-24a, whereinQ₂ is a bond

26a. Insulin derivative according to any of paragraphs 21a-25a, whereinX₁ is —C═O

27a. Insulin derivative according to any of paragraphs 20a-26a, whereinQ₂ is selected from the group consisting of CO(CONH₂)CH—;—(CO—(CH₂)₂₋₆—NH—CO)₁₋₄—; —(CO—(CH₂)₂₋₆—CO—NH)₁₋₄—;

28a. Insulin derivative according to any of paragraphs 20a or 27a,wherein Q₂ is selected from the group consisting of—(CO—(CH₂)₂—NH—CO)₁—-(CO—(CH₂)₃—NH—CO)₁—, —(CO—(CH₂)₄—NH—CO)₁— or—(CO—(CH₂)₅—NH—CO)₁—.

29a. Insulin derivative according to paragraphs 27a or 28a, wherein Q₁is a bond.

30a. Insulin derivative according to any of paragraphs 20a-29a, whereinQ₃ is —(CH₂)_(m)— where m is an integer in the range of 6 to 32 or from8 to 20.

31a. Insulin derivative according to paragraphs 30a, where m is 12, 13,14, 15 or 16.

32a. Insulin derivative according to any of paragraphs 20a-31a, whereinZ is —COOH.

33a. Insulin derivative according to any of paragraphs 20a-31a, whereinZ is —CH(COOH)₂.

34a. An insulin derivative according to any of paragraphs 20a-31a,wherein Z is —N(CH₂COOH)₂.

35a. An insulin derivative according to any of paragraphs 20a-31a,wherein Z is —SO₃H.

36a. An insulin derivative according to any of paragraphs 20a-31a,wherein Z is —PO₃H.

37a. Insulin derivative according to any of the preceeding paragraphsselected from the group consisting ofN^(εB29)-ω-carboxy-pentadecanoyl-γ-L-glutamylamide desB30 human insulin,N^(εB29)-ω-carboxy-pentadecanoyl-γ-amino-butanoyl desB30 human insulin,N^(εB29)-ω-carboxy-tetradecanoyl-γ-L-glutamylamide desB30 human insulin,N^(εB29)-ω-carboxy-tridecanoyl-γ-L-glutamylamide desB30 human insulin,N^(εB29)-ω-carboxy-pentadecanoyl-β-alanyl desB30 human insulin,N^(εB29)-ω-carboxy-pentadecanoyl-γ-L-aspartylamide desB30 human insulin,N^(εB29)-ω-carboxy-pentadecanoyl-ε-aminohexanoyl desB30 human insulin,N^(εB29)-ω-carboxy-pentadecanoyl-δ-aminopentanoyl desB30 human insulin.

38a. Insulin derivative according to paragraphs 11a, wherein

Q₁ is:

-   -   an α-amino acid amide residue having a carboxylic acid group in        the substituent which residue forms, with one of its carboxylic        acid groups, an amide group together with the α-amino group of        the N-terminal amino acid residue of the B chain or together        with the ε-amino group of a Lys residue present in the B chain        of the parent insulin;    -   a chain composed of two, three or four α-amino acid amide        residues linked together via amide bonds, which chain—via an        amide bond—is linked to the α-amino group of the N-terminal        amino acid residue of the B chain or to the ε-amino group of a        Lys residue present in the B chain of the parent insulin, the        amino acid residues of W being selected from the group of amino        acid residues having a neutral substituent and amino acid        residues having a carboxylic acid group in the substituent so        that W has at least one amino acid residue which has a        carboxylic acid group in the substituent; or    -   a bond

Q₂ is:

—COCH(CONH₂)—

—COCH₂N(CH₂CONH₂)—

—COCH₂N(CH₂CONH₂)COCH₂N(CH₂CONH₂)

—COCH₂CH₂N(CH₂CH₂CONH₂)—

—COCH₂CH₂N(CH₂CH₂CONH₂)—COCH₂CH₂N(CH₂CH₂CONH₂)—

—COCH₂N(CH₂CH₂CONH₂)—

—COCH₂CH₂N(CH₂CONH₂)—

—(CO—(CH₂)₂₋₆—NH—CO)₁₋₄—;

—(CO—(CH₂)₂₋₆—CO—NH)₁₋₄—;

—(CO—(CR₉R₁₀)₁₋₆—CO—NH)₁₋₄—, where R₉ and R₁₀, independently of eachother can be H, —CH₃, —(CH₂)₁₋₆CH₃ or —CONH₂; or

a bond

-   -   provided that at least one of Q₁ or Q₂ is not a bond;    -   Q₃ is    -   —((CR₁R₂)₁₋₆—NR₁₅—CO)₁₋₄—, where R₁, R₂ and R₁₅ independently of        each other can be H, —CH₃, —CH₁₋₆CH₃, —SO₃H, —(CH₂)₁₋₆—SO₃H,        —(CH₂)₁₋₆—O—PO₃H₂ or CONH₂ and R₁₅ can be arylene which may be        substituted with one or two groups of R₁, R₂ as defined above;    -   NR₁₅ where R₁₅ is defined as above,    -   a bond

Q₄ is

-   -   —(CH₂)_(m)— where m is an integer from 4 to 22;    -   a divalent hydrocarbon chain comprising 1, 2 or 3 —CH═CH— groups        and a number of —CH₂— groups sufficient to give a total number        of carbon atoms in the chain in the range of 4 to 22;    -   arylene or heteroarylene, which may be substituted with one or        two groups selected from the group consisting of —COOH, —CH₃,        —CH_(p)CH₃, —SO₃H, —(CH₂)_(p)—SO₃H, —CONR₁R₂ or —SO₂NR₁R₂, where        R₁ and R₂, independently of each other can be H, —CH₃,        —(CH)₁₋₆—CH₃, —SO₃H, —(CH₂)₁₋₆—SO₃H, —(CH₂)_(p)—O—PO₃H₂ or        CONH₂; or    -   a divalent hydrocarbon chain of the formula

—(CH₂)_(s)—Y₁—(C₆H₄)_(v1)—Y₂—(CH₂)_(w)—Y₃—(C₆H₄)_(v2)—Y₄—(CH₂)_(t)—Y₅—(C₆H₄)_(v3)—Y₆—(CH₂)_(z)—

-   -   wherein Y₁-Y₆ independently of each other can be O; S or a bond;        where s, w, t and z independently of each other are zero or an        integer from 1 to 10 so that the sum of s, w, t and z is in the        range from 4 to 30, and v₁, v₂, and v₃ independently of each        other can be zero or 1 with the proviso that Y₁-Y₆ do not link        to each other; or X₁ is

O;

—C═O

a bond; or

-   -   where R is hydrogen, C₁₋₃-alkyl, C₂₋₃-alkenyl or C₂₋₃-alkynyl;        and X₂, X₃ and Q₅ are bonds;        All values of n are zero; and

Z is:

-   -   —COOH;    -   —CO-Asp;    -   —CO-Glu;    -   —CO-Gly;    -   —CO-Sar;    -   —CH(COOH)₂;    -   —N(CH₂COOH)₂;    -   —SO₃H    -   —OSO₃H    -   —OPO3H₂    -   —PO₃H₂ or    -   -tetrazol-5-yl or    -   —O—W₁,    -   where W₁ is arylene or heteroarylene, which may be substituted        with one or two groups selected from the group consisting of        —COOH, —SO₃H, —(CH₂)₁₋₆—SO₃H, —CONR₁₃R₁₄ or —SO₂NR₁₂R₁₄, where        R₁₃ and R₁₄, independently of each other can be H, —SO₃H,        —(CH₂)₁₋₆—SO₃H, —(CH₂)₁₋₆—O—PO₃H₂, —CONH₂ or tetrazo-5-lyl;        and any Zn²⁺ complex thereof.

39a. Insulin derivative according to paragraphs 38a, wherein Q₁ is anamino acid amide residue having from 4 to 10 carbon atoms.

40a. Insulin derivative according to any of paragraphs 38a-39a, whereinQ₁ is selected from the group consisting of β-D-Asp-amide,β-L-Asp-amide, γ-L-Glu-amide and γ-D-Glu-amide.

41a. Insulin derivative according to paragraphs 38a, wherein Q₁ is achain of amino acid amide residues.

42a. Insulin derivative according to paragraphs 38a or 41a, wherein Q₁is a chain of two amino acid amide residues selected from the groupconsisting of β-L-Asp-amide-β-L-Asp-amide, β-L-Asp-amide-γ-L-Glu-amide,γ-L-Glu-amide-γ-L-Glu-amide, γ-L-Glu-amide-β-L-Asp-amide,β-L-Asp-amide-β-D-Asp-amide, β-L-Asp-amide-γ-D-Glu-amide,γ-L-Glu-amide-γ-D-Glu-amide, γ-L-Glu-amide-β-D-Asp-amide,β-D-Asp-amide-β-L-Asp-amide, β-D-Asp-amide-γ-L-Glu-amide,γ-D-Glu-amide-γ-L-Glu-amide, γ-D-Glu-amide-β-L-Asp-amide,β-D-Asp-amide-β-D-Asp-amide, β-D-Asp-amide-γ-D-Glu-amide,γ-D-Glu-amide-γ-D-Glu-amide, γ-D-Glu-amide-β-D-Asp-amide,

43a Insulin derivative according to any of paragraphs 38a-42a, whereinQ₂ is a bond

44a. Insulin derivative according to any of paragraphs 38a-42a, whereinQ₂ is selected from the group consisting of CO(CONH₂)CH—;—(CO—(CH₂)₂₋₆—NH—CO)₁₋₄— and —(CO—(CH₂)₂₋₆—CO—NH)₁₋₄—.

45a. Insulin derivative according to paragraphs 44a, wherein Q₁ is abond.

46a. Insulin derivative according to any of paragraphs 38a, 44a or 45a,wherein Q₂ is selected from the group consisting of —(CO—(CH₂)₂—NH—CO)₁—or —(CO—(CH₂)₃—NH—CO)₁₋₄—

47a. Insulin derivative according to paragraphs 37a, wherein Q₄ is—(CH₂)_(s)—Y₁—(C₆H₄)_(v1)—Y₂—(CH₂)_(w)—Y₃—(C₆H₄)_(v2)—Y₄—(CH₂)_(t)—Y₅—(C₆H₄)_(v3)—Y₆—(CH₂)_(z)—

wherein Y₁-Y₆ independently of each other can be O; S or a bond; wheres, w, t and z independently of each other are zero or an integer from 1to 10 so that the sum of s, w, t and z is in the range from 4 to 30, andv₁, v₂, and v₃ independently of each other can be zero or 1 with theproviso that Y₁-Y₆ do not link to each other.

48a. Insulin derivative according to any of paragraphs 37a or 46a,wherein at least two of v₁, v₂, or v₃ are zero.

49a. Insulin derivative according to any of paragraphs 38a, 47a or 48a,wherein at Y₁—Y₆ are bonds.

50a. Insulin derivative according to any of paragraphs 38a, 47a or 48a,wherein at least one of Y₁-Y₆ are O or S.

51a. Insulin derivative according to any of paragraphs 38a or 47a-48a,wherein Y₁ is O or S and v₁ is one.

52a. Insulin derivative according to any of paragraphs 38a-51a, whereinZ is —COOH.

53a. Insulin derivative according to any of paragraphs 38a-51a, whereinZ is —CH(COOH)₂.

54a. An insulin derivative according to any of paragraphs 38a-51a,wherein Z is —N(CH₂COOH)₂.

55a. An insulin derivative according to any of paragraphs 38a-51a,wherein Z is —SO₃H.

56a. An insulin derivative according to any of paragraphs 38a-51a,wherein Z is —PO₃H.

57a. An insulin derivative according to any of paragraphs 38a-51a,wherein Z is —O—W₁,

where W₁ is arylene or heteroarylene, which may be substituted with oneor two groups selected from the group consisting of —COOH, —SO₃H,—(CH₂)₁₋₆—SO₃H, —CONR₁₃R₁₄ or —SO₂NR₁₂R₁₄, where R₁₃ and R₁₄,independently of each other can be H, —SO₃H, —(CH₂)₁₋₆—SO₃H,—(CH₂)₁₋₆—O—PO₃H₂, —CONH₂ or tetrazo-5-lyl.

58a. Insulin derivative according to any of paragraphs 1a-19a or 38a-57awherein the insulin derivative are selected from the group consisting ofN^(εB29)-10-(4-carboxyphenoxy)-decanoyl-γ-L-glutamylamide desB30 humaninsulin, N^(εB29)-4-[11-(4-Carboxyphenyl)undecanoylamino]butyryl desB30human insulin.

59a. Insulin derivative according to paragraphs 11a, wherein

Q₁ is:

-   -   an α-amino acid amide residue having a carboxylic acid group in        the substituent which residue forms, with one of its carboxylic        acid groups, an amide group together with the α-amino group of        the N-terminal amino acid residue of the B chain or together        with the ε-amino group of a Lys residue present in the B chain        of the parent insulin;    -   a chain composed of two, three or four α-amino acid amide        residues linked together via amide bonds, which chain—via an        amide bond—is linked to the α-amino group of the N-terminal        amino acid residue of the B chain or to the ε-amino group of a        Lys residue present in the B chain of the parent insulin, the        amino acid residues of W being selected from the group of amino        acid residues having a neutral substituent and amino acid        residues having a carboxylic acid group in the substituent so        that W has at least one amino acid residue which has a        carboxylic acid group in the substituent; or    -   a bond

Q₂ is:

—COCH(CONH₂)—

—COCH₂N(CH₂CONH₂)—

—COCH₂N(CH₂CONH₂)COCH₂N(CH₂CONH₂)

—COCH₂CH₂N(CH₂CH₂CONH₂)—

—COCH₂CH₂N(CH₂CH₂CONH₂)—COCH₂CH₂N(CH₂CH₂CONH₂)—

—COCH₂N(CH₂CH₂CONH₂)—

—COCH₂CH₂N(CH₂CONH₂)—

—(CO—(CH₂)₂₋₆—NH—CO)₁₋₄—;

—(CO—(CH₂)₂₋₆—CO—NH)₁₋₄—;

—(CO—(CR₉R₁₀)₁₋₆—CO—NH)₁₋₄—, where R₉ and R₁₀, independently of eachother can be H, —CH₃, —(CH₂)₁₋₆CH₃ or —CONH₂; or

a bond

-   -   provided that at least one of Q₁ or Q₂ is not a bond;        n is independently 2 or 3;        Q₃, Q₄, and Q₅ independently of each other can be    -   (CH₂CH₂O)_(y)—; (CH₂CH₂CH₂O)_(y)—; (CH₂CH₂CH₂CH₂O)_(y)—;        (CH₂CH₂OCH₂CH₂CH₂CH₂O)_(y)— or (CH₂CH₂CH₂OCH₂CH₂CH₂CH₂O)_(y)—;        —(CH₂OCH₂)_(y)— where y is 1-20;    -   —(CH₂)_(m)— where m is an integer in the range of 1 to 32;    -   a divalent hydrocarbon chain comprising 1, 2 or 3 —CH═CH— groups        and a number of —CH₂— groups sufficient to give a total number        of carbon atoms in the chain in the range of 4 to 32;    -   —(CR₃R₄)₁₋₆-(NHCO—(CR₃R₄)₁₋₆—NHCO)₁₋₂—(CR₃R₄)₁₋₆ or        —(CR₃R₄)₁₋₆—(CONH—(CR₃R₄)₁₋₆—CONH)₁₋₂—(CR₃R₄)₁₋₆—,        —(CR₃R₄)₁₋₆—(NHCO—(CR₃R₄)₁₋₆—CONH)₁₋₂—(CR₃R₄)₁₋₆— or        —(CR₃R₄)₁₋₆—(CONH—(CR₃R₄)₁₋₆—NHCO)₁₋₂—(CR₃R₄)₁₋₆ where R₃ and R₄        independently of each other and independently for each carbon        can be H, —COOH or OH,    -   —(CR₅R₆)₁₋₆—, where R₅ and R₆ independently of each other and        independently for each carbon can be H, —COOH, (CH₂)₁₋₆COOH; or    -   a bond;        -   with the proviso that Q₃-Q₅ are different;            X₁, X₂ and X₃ are independently

O;

—C═O;

a bond; or

-   -   where R is hydrogen, C₁₋₃-alkyl, C₂₋₃-alkenyl or C₂₋₃-alkynyl;        and    -   Z is:    -   —COOH;    -   —CO-Asp;    -   —CO-Glu;    -   —CO-Gly;    -   —CO-Sar;    -   —CH(COOH)₂;    -   —N(CH₂COOH)₂;    -   —SO₃H    -   —OSO₃H    -   —OPO3H₂    -   —PO₃H₂ or    -   -tetrazol-5-yl        and any Zn²⁺ complex thereof.

60a. Insulin derivative according to paragraphs 59a, wherein Q₁ is anamino acid amide residue having from 4 to 10 carbon atoms.

61a. Insulin derivative according to any of paragraphs 59a-60a, whereinQ₁ is selected from the group consisting of β-D-Asp-amide,β-L-Asp-amide, γ-L-Glu-amide and γ-D-Glu-amide.

62a. Insulin derivative according to paragraphs 59a, wherein Q₁ is achain of amino acid amide residues.

63a. Insulin derivative according to paragraphs 59a or 62a, wherein Q₁is a chain of two amino acid amide residues selected from the groupconsisting of β-L-Asp-amide-β-L-Asp-amide, β-L-Asp-amide-γ-L-Glu-amide,γ-L-Glu-amide-γ-L-Glu-amide, γ-L-Glu-amide-β-L-Asp-amide,β-L-Asp-amide-β-D-Asp-amide, β-L-Asp-amide-γ-D-Glu-amide,γ-L-Glu-amide-γ-D-Glu-amide, γ-L-Glu-amide-β-D-Asp-amide,β-D-Asp-amide-β-L-Asp-amide, β-D-Asp-amide-γ-L-Glu-amide,γ-D-Glu-amide-γ-L-Glu-amide, γ-D-Glu-amide-β-L-Asp-amide,β-D-Asp-amide-β-D-Asp-amide, β-D-Asp-amide-γ-D-Glu-amide,y-D-Glu-amide-γ-D-Glu-amide, γ-D-Glu-amide-β-D-Asp-amide,

64a. Insulin derivative according to any of paragraphs 59a-63a, whereinQ₂ is a bond

65a. Insulin derivative according to any of paragraphs 59a-63a, whereinQ₂ is selected from the group consisting of CO(CONH₂)CH—;—(CO—(CH₂)₂₋₆—NH—CO)₁₋₄—; —(CO—(CH₂)₂₋₆—CO—NH)₁₋₄—;

66a. Insulin derivative according to any of paragraphs 59a or 65a,wherein Q₂ is selected from the group consisting of —(CO—(CH₂)₂—NH—CO)₁—or —(CO—(CH₂)₃—NH—CO)₁₋₄—

67a. Insulin derivative according to paragraphs 65a or 66a, wherein Q₁is a bond.

68a. Insulin derivative according any of paragraphs 59a-67a, wherein oneof Q₃, Q₄, or Q₅ is —(CH₂)_(m)— where m is an integer in the range of 1to 32 or 1-12.

69a. Insulin derivative according to any of paragraphs 59a-67a, whereinone of Q₃, Q₄, or Q₅ is (CH₂CH₂O)_(y)—; (CH₂CH₂CH₂O)_(y)—;(CH₂CH₂CH₂CH₂O)_(y)—; (CH₂CH₂OCH₂CH₂CH₂CH₂O)_(y)— or(CH₂CH₂CH₂OCH₂CH₂CH₂CH₂O)_(y)—; —(CH₂OCH₂)_(y)— where y is 1-20;

70a. Insulin derivative according to paragraphs 69a, wherein one of Q₃,Q₄, or Q₅ is (CH₂CH₂O)_(y)— or (CH₂CH₂OCH₂CH₂CH₂CH₂O) wherein y is inthe range of 2-12, 2-4 or 2-3

71a. Insulin derivative according to any of paragraphs 59a, 69a or 70a,wherein y is 1.

72a. Insulin derivative according to any of paragraphs 59a-71a, whereinZ is —COOH.

73a. Insulin derivative according to any of paragraphs 59a-71a, whereinZ is —CH(COOH)₂.

74a. An insulin derivative according to any of paragraphs 59a-71a,wherein Z is —N(CH₂COOH)₂.

75a. An insulin derivative according to any of paragraphs 59a-71a,wherein Z is —SO₃H.

76a. An insulin derivative according to any of paragraphs 59a-71a,wherein Z is —PO₃H.

77a. Insulin derivative according to any of paragraphs 1a-19a or 59a-76aselected from the group consisting of

-   N^(εB29)-(3-(3-{4-[3-(7-carboxyheptanoylamino)propoxy]butoxy}propylcarbamoyl)-propionyl-y-glutamylamide)    desB30 human Insulin

78a. Insulin derivative according to any of the paragraphs 1a-77a,wherein the parent insulin is human insulin or porcine insulin

79a Insulin derivative according to any of the paragraphs 1a77a, whereinthe parent insulin is an insulin analogue.

80a. Insulin derivative according to any of paragraphs 78a-79a, whereinthe amino acid residue at position B30 of the parent insulin is Lys orhas been deleted.

81a. Insulin derivative according to paragraphs 80a, wherein the parentinsulin is desB30 human insulin.

82a. Insulin derivative according to any of paragraphs 78a-81a, whereinthe amino acid residue at position B1 of the parent insulin has beendeleted.

83a. Insulin derivative according to any of paragraphs 78a-82a, whereinthe amino acid residue in position A21 of the parent insulin is Gly orAsn.

84a. Insulin derivative according to any of paragraphs 78a-83a, whereinthe amino acid residue at position B3 of the parent insulin is Lys

85a. Insulin derivative according to any of paragraphs 78a-84a, whereinthe amino acid residue at position B28 of the parent insulin is Asp orLys.

86a. Insulin derivative according to any of paragraphs 78a-85a, whereinthe amino acid residue at position B29 of the parent insulin is Pro orThr.

87a. Insulin derivative according to paragraphs 85a, wherein the parentinsulin is AspB28 human insulin

88a. Insulin derivative according to paragraphs 83a, wherein the parentinsulin is GlyA21 human insulin or GlyA21desB30 human insulin orGlyA21ArgB31ArgB32 human insulin.

89a. Insulin derivative according to paragraphs 84a, wherein the parentinsulin is LysB3GluB29 human insulin.

90a. Insulin derivative according to paragraphs 85a-86a, wherein theparent insulin is LysB28ProB29 human insulin

91a. Insulin derivative according to paragraphs 80a and 86a, wherein theparent insulin is ThrB29LysB30 human insulin

92a. A zinc complex of an insulin derivative according to any of thepreceding paragraphs wherein each insulin hexamer binds two zinc ions,three zinc ions or four zinc ions.

93a. A pharmaceutical composition for the treatment of diabetes in apatient in need of such treatment, comprising a therapeuticallyeffective amount of an insulin derivative according to any of thepreceding paragraphs together with a pharmaceutically acceptablecarrier.

94a. A pharmaceutical composition for the treatment of diabetes in apatient in need of such treatment, comprising a therapeuticallyeffective amount of an insulin derivative according to any of thepreceding paragraphs in mixture with an insulin or an insulin analoguewhich has a rapid onset of action, together with a pharmaceuticallyacceptable carrier.

95a. A method of treating diabetes in a patient in need of such atreatment, comprising administering to the patient a therapeuticallyeffective amount of an insulin derivative according to paragraphs 1a-94atogether with a pharmaceutically acceptable carrier.

94a. A method of treating diabetes in a patient in need of such atreatment, comprising administering to the patient a therapeuticallyeffective amount of an insulin derivative according to paragraphs 1a-94ain mixture with an insulin or an insulin analogue which has a rapidonset of action, together with a pharmaceutically acceptable carrier.

97a. A method according to paragraphs 95a or 96a for pulmonary treatmentof diabetes

98a. A mixture of an insulin derivative according to any of paragraphs1a-92a and a rapid acting insulin analogue selected group consisting ofAspB28 human insulin; LysB28ProB29 human insulin and LysB3GluB29 humaninsulin.

99a. Insulin derivative as described in the examples.

The starting product for the acylation, the parent insulin or insulinanalogue or a precursor thereof can be produced by either well-knowpeptide synthesis or by well known recombinant production in suitabletransformed microorganisms. Thus the insulin starting product can beproduced by a method which comprises culturing a host cell containing aDNA sequence encoding the polypeptide and capable of expressing thepolypeptide in a suitable nutrient medium under conditions permittingthe expression of the peptide, after which the resulting peptide isrecovered from the culture.

The medium used to culture the cells may be any conventional mediumsuitable for growing the host cells, such as minimal or complex mediacontaining appropriate supplements. Suitable media are available fromcommercial suppliers or may be prepared according to published recipes(e.g. in catalogues of the American Type Culture Collection). Thepeptide produced by the cells may then be recovered from the culturemedium by conventional procedures including separating the host cellsfrom the medium by centrifugation or filtration, precipitating theproteinaceous components of the supernatant or filtrate by means of asalt, e.g. ammonium sulphate, purification by a variety ofchromatographic procedures, e.g. ion exchange chromatography, gelfiltration chromatography, affinity chromatography, or the like,dependent on the type of peptide in question.

The DNA sequence encoding the parent insulin may suitably be of genomicor cDNA origin, for instance obtained by preparing a genomic or cDNAlibrary and screening for DNA sequences coding for all or part of thepolypeptide by hybridisation using synthetic oligonucleotide probes inaccordance with standard techniques (see, for example, Sambrook, J.Fritsch, E F and Maniatis, T. Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratory Press, New York, 1989). The DNA sequenceencoding the parent insulin may also be prepared synthetically byestablished standard methods, e.g. the phosphoamidite method describedby Beaucage and Caruthers, Tetrahedron Letters 22 (1981), 1859-1869, orthe method described by Matthes et al., EMBO Journal 3 (1984), 801-805.The DNA sequence may also be prepared by polymerase chain reaction usingspecific primers, for instance as described in U.S. Pat. No. 4,683,202or Saiki et al., Science 239 (1988), 487-491.

The DNA sequence may be inserted into any vector which may convenientlybe subjected to recombinant DNA procedures, and the choice of vectorwill often depend on the host cell into which it is to be introduced.Thus, the vector may be an autonomously replicating vector, i.e. avector which exists as an extrachromosomal entity, the replication ofwhich is independent of chromosomal replication, e.g. a plasmid.Alternatively, the vector may be one which, when introduced into a hostcell, is integrated into the host cell genome and replicated togetherwith the chromosome(s) into which it has been integrated.

The vector is for example an expression vector in which the DNA sequenceencoding the parent insulin is operably linked to additional segmentsrequired for transcription of the DNA, such as a promoter. The promotermay be any DNA sequence which shows transcriptional activity in the hostcell of choice and may be derived from genes encoding proteins eitherhomologous or heterologous to the host cell. Examples of suitablepromoters for directing the transcription of the DNA encoding the parentinsulin in a variety of host cells are well known in the art, cf. forinstance Sambrook et al., supra.

The DNA sequence encoding the parent insulin may also, if necessary, beoperably connected to a suitable terminator, polyadenylation signals,transcriptional enhancer sequences, and translational enhancersequences. The recombinant vector of the invention may further comprisea DNA sequence enabling the vector to replicate in the host cell inquestion.

The vector may also comprise a selectable marker, e.g. a gene theproduct of which complements a defect in the host cell or one whichconfers resistance to a drug, e.g. ampicillin, kanamycin, tetracyclin,chloramphenicol, neomycin, hygromycin or methotrexate.

To direct a peptide of the present invention into the secretory pathwayof the host cells, a secretory signal sequence (also known as a leadersequence, prepro sequence or pre sequence) may be provided in therecombinant vector. The secretory signal sequence is joined to the DNAsequence encoding the peptide in the correct reading frame. Secretorysignal sequences are commonly positioned 5′ to the DNA sequence encodingthe peptide. The secretory signal sequence may be that normallyassociated with the peptide or may be from a gene encoding anothersecreted protein.

The procedures used to ligate the DNA sequences coding for the parentinsulin, the promoter and optionally the terminator and/or secretorysignal sequence, respectively, and to insert them into suitable vectorscontaining the information necessary for replication, are well known topersons skilled in the art (cf., for instance, Sambrook et al., supra).

The host cell into which the DNA sequence or the recombinant vector isintroduced may be any cell which is capable of producing the presentpeptide and includes bacteria, yeast, fungi and higher eukaryotic cells.Examples of suitable host cells well known and used in the art are,without limitation, E. coli, Saccharomyces cerevisiae, or mammalian BHKor CHO cell lines.

The parent insulin molecule is then converted into the insulinderivatives of the invention by introducing of the relevant substituentin either the B1 position or in the chosen Lys position in the B-chain.The substituent can be introduced by any convenient method and manymethods are disclosed in the prior art for acylation of an amino group.More details will appear from the following examples.

Insulin derivatives according to the invention may be provided in theform of essentially zinc free compounds or in the form of zinccomplexes. When zinc complexes of an insulin derivative according to theinvention are provided, two Zn²⁺ ions, three Zn²⁺ ions, four Zn²⁺ ions,five Zn²⁺ ions, six Zn²⁺ ions, seven Zn²⁺ ions, eight Zn²⁺ ions, nineZn²⁺ ions or ten Zn²⁺ ions can be bound per six molecules of insulinderivative. Solutions of zinc complexes of the insulin derivatives willcontain mixtures of such species.

In one aspect the invention is related to a pharmaceutical compositioncomprising a therapeutically effective amount of an insulin derivativeor a zinc complex of the insulin derivative according to the inventionoptionally together with a pharmaceutically acceptable carrier and/or apharmaceutically acceptable additive, which composition can be providedfor the treatment of type 1 diabetes, type 2 diabetes and other statesthat cause hyperglycaemia in patients in need of such a treatment.

In one aspect of the invention, there is provided a method of treatingtype 1 diabetes, type 2 diabetes and other states that causehyperglycaemia in a patient in need of such a treatment, comprisingadministering to the patient a therapeutically effective amount of anpharmaceutical composition comprising the insulin derivative or a zinccomplex of the insulin derivative according to the invention optionallytogether with a pharmaceutically acceptable carrier and/orpharmaceutical acceptable additives.

In one aspect of the invention, there is provided a method for themanufacture of a pharmaceutical composition for the use in the treatmentof type 1 diabetes, type 2 diabetes and other states that causehyperglycaemia, the composition comprising an insulin derivative or azinc complex of the insulin derivative according to the inventionoptionally together with a pharmaceutically acceptable carrier and/orpharmaceutical acceptable additives.

In one aspect of the invention, there is provided a pharmaceuticalcomposition for treating type 1 diabetes, type 2 diabetes and otherstates that cause hyperglycaemia in a patient in need of such atreatment, the composition comprising a therapeutically effective amountof an insulin derivative or a zinc complex of the insulin derivativeaccording to the invention in mixture with an insulin or an insulinanalogue which has a rapid onset of action, optionally together withpharmaceutically acceptable carriers and/or additives.

In one aspect of the invention, there is provided a method of treatingtype 1 diabetes, type 2 diabetes and other states that causehyperglycaemia in a patient in need of such a treatment, comprisingadministering to the patient a therapeutically effective amount of anpharmaceutical composition comprising the insulin derivative or a zinccomplex of the insulin derivative according to the invention in mixturewith an insulin or an insulin analogue which has a rapid onset ofaction, optionally together with a pharmaceutically acceptable carrierand/or pharmaceutical acceptable additives.

In one aspect of the invention, there is provided a method for themanufacture of a pharmaceutical composition for the use in the treatmentof type 1 diabetes, type 2 diabetes and other states that causehyperglycaemia, the composition comprising a therapeutically effectiveamount of an insulin derivative or a zinc complex of the insulinderivative according to the invention in mixture with an insulin or aninsulin analogue which has a rapid onset of action, optionally togetherwith a pharmaceutically acceptable carrier and/or pharmaceuticalacceptable additives.

In one aspect the invention provides a pharmaceutical composition beinga mixture of an insulin derivative or a zinc complex of the insulinderivative according to the invention and a rapid acting insulinanalogue selected group consisting of AspB28 human insulin; LysB28ProB29human insulin and LysB3GluB29 human insulin.

One aspect of the invention is related to a pharmaceutical compositioncomprising a therapeutically effective amount of an insulin derivativeor a zinc complex of the insulin derivative according to the inventionoptionally together with a pharmaceutically acceptable carrier and/or apharmaceutically acceptable additive, which can be provided forpulmonary treatment of type 1 diabetes, type 2 diabetes and other statesthat cause hyperglycaemia in patients in need of such a treatment.

In one aspect the invention is related to application of apharmaceutical composition for pulmonary treatment of type 1 diabetes,type 2 diabetes and other states that cause hyperglycaemia in a patientin need of such a treatment, the pharmaceutical composition comprising atherapeutically effective amount of an insulin derivative or a zinccomplex of the insulin derivative according to the invention optionallyin mixture with an insulin or an insulin analogue which has a rapidonset of action, and optionally together with pharmaceuticallyacceptable carriers and/or additives.

In one aspect of the invention, there is provided a method for themanufacture of a pharmaceutical composition for the use in the treatmentof type 1 diabetes, type 2 diabetes and other states that causehyperglycaemia, the composition being used pulmonary and comprising atherapeutically effective amount of an insulin derivative or a zinccomplex of the insulin derivative according to the invention optionallyin mixture with an insulin or an insulin analogue which has a rapidonset of action, and optionally together with a pharmaceuticallyacceptable carrier and/or pharmaceutical acceptable additives.

The insulin derivative according to the invention and the rapid actinginsulin analogue can be mixed in a ratio from about 90/10%; about 70/30%or about 50/50%.

In one aspect, the invention relates to a pharmaceutical compositioncomprising an insulin derivative according to the invention which issoluble at physiological pH values.

In one aspect, the invention relates to a pharmaceutical compositioncomprising an insulin derivative according to the invention which issoluble at pH values in the interval from about 6.5 to about 8.5.

In one aspect, the invention relates to a pharmaceutical compositionwith a prolonged profile of action which comprises an insulin derivativeaccording to the invention.

In one aspect, the invention relates to a pharmaceutical compositionwhich is a solution containing from about 120 nmol/ml to about 2400nmol/ml, from about 400 nmol/ml to about 2400 nmol/ml, from about 400nmol/ml to about 1200 nmol/ml, from about 600 nmol/ml to about 2400nmol/ml, or from about 600 nmol/ml to about 1200 nmol/ml of an insulinderivative according to the invention or of a mixture of the insulinderivative according to the invention with a rapid acting insulinanalogue.

Pharmaceutical Compositions

The insulin derivatives of this invention of the claimed formula can,for example, be administered subcutaneously, orally, or pulmonary.

For subcutaneous administration, the compounds of the formula areformulated analogously with the formulation of known insulins.Furthermore, for subcutaneous administration, the compounds of theformula are administered analogously with the administration of knowninsulins and, generally, the physicians are familiar with thisprocedure.

The insulin derivatives of this invention may be administered byinhalation in a dose effective manner to increase circulating insulinlevels and/or to lower circulating glucose levels. Such administrationcan be effective for treating disorders such as diabetes orhyperglycemia. Achieving effective doses of insulin requiresadministration of an inhaled dose of insulin derivative of thisinvention of more than about 0.5 μg/kg to about 50 μg/kg. Atherapeutically effective amount can be determined by a knowledgeablepractitioner, who will take into account factors including insulinlevel, blood glucose levels, the physical condition of the patient, thepatient's pulmonary status, or the like.

Administration by inhalation can result in pharmacokinetics comparableto subcutaneous administration of insulins. Different inhalation devicestypically provide similar pharmacokinetics when similar particle sizesand similar levels of lung deposition are compared.

According to the invention, insulin derivative of this invention may bedelivered by any of a variety of inhalation devices known in the art foradministration of a therapeutic agent by inhalation. These devicesinclude metered dose inhalers, nebulizers, dry powder generators,sprayers, and the like. Insulin derivative of this invention isdelivered by a dry powder inhaler or a sprayer. There are a severaldesirable features of an inhalation device for administering insulinderivative of this invention. For example, delivery by the inhalationdevice is advantageously reliable, reproducible, and accurate. Theinhalation device should deliver small particles, for example, less thanabout 10 μm, for example about 1-5 μm, for good respirability. Somespecific examples of commercially available inhalation devices suitablefor the practice of this invention are Turbohaler™ (Astra), Rotahaler®(Glaxo), Diskus® (Glaxo), Spiros™ inhaler (Dura), devices marketed byInhale Therapeutics, AERx™ (Aradigm), the Ultravent® nebulizer(Mallinckrodt), the Acorn II® nebulizer (Marquest Medical Products), theVentolin® metered dose inhaler (Glaxo), the Spinhaler® powder inhaler(Fisons), or the like.

As those skilled in the art will recognize, the formulation of insulinderivative of this invention, the quantity of the formulation delivered,and the duration of administration of a single dose depend on the typeof inhalation device employed. For some aerosol delivery systems, suchas nebulizers, the frequency of administration and length of time forwhich the system is activated will depend mainly on the concentration ofinsulin conjugate in the aerosol. For example, shorter periods ofadministration can be used at higher concentrations of insulin conjugatein the nebulizer solution. Devices such as metered dose inhalers canproduce higher aerosol concentrations, and can be operated for shorterperiods to deliver the desired amount of insulin conjugate. Devices suchas powder inhalers deliver active agent until a given charge of agent isexpelled from the device. In this type of inhaler, the amount of insulinderivative of this invention in a given quantity of the powderdetermines the dose delivered in a single administration.

The particle size of insulin derivative of this invention in theformulation delivered by the inhalation device is critical with respectto the ability of insulin to make it into the lungs, and into the lowerairways or alveoli. The insulin derivative of this invention can beformulated so that at least about 10% of the insulin conjugate deliveredis deposited in the lung, for example about 10 to about 20%, or more. Itis known that the maximum efficiency of pulmonary deposition for mouthbreathing humans is obtained with particle sizes of about 2 μm to about3 μm. When particle sizes are above about 5 μm pulmonary depositiondecreases substantially. Particle sizes below about 1 μm cause pulmonarydeposition to decrease, and it becomes difficult to deliver particleswith sufficient mass to be therapeutically effective. Thus, particles ofthe insulin derivative delivered by inhalation have a particle size lessthan about 10 μm, for example in the range of about 1 μm to about 5 μm.The formulation of the insulin derivative is selected to yield thedesired particle size in the chosen inhalation device.

Advantageously for administration as a dry powder, an insulin derivativeof this invention is prepared in a particulate form with a particle sizeof less than about 10 μm, for example about 1 to about 5 μm. Theparticle size is effective for delivery to the alveoli of the patient'slung. The dry powder is largely composed of particles produced so that amajority of the particles have a size in the desired range.Advantageously, at least about 50% of the dry powder is made ofparticles having a diameter less than about 10 μm. Such formulations canbe achieved by spray drying, milling, or critical point condensation ofa solution containing insulin conjugate and other desired ingredients.Other methods also suitable for generating particles useful in thecurrent invention are known in the art.

The particles are usually separated from a dry powder formulation in acontainer and then transported into the lung of a patient via a carrierair stream. Typically, in current dry powder inhalers, the force forbreaking up the solid is provided solely by the patient's inhalation. Inanother type of inhaler, air flow generated by the patient's inhalationactivates an impeller motor which deagglomerates the particles.

Formulations of insulin derivatives of this invention for administrationfrom a dry powder inhaler typically include a finely divided dry powdercontaining the derivative, but the powder can also include a bulkingagent, carrier, excipient, another additive, or the like. Additives canbe included in a dry powder formulation of insulin conjugate, forexample, to dilute the powder as required for delivery from theparticular powder inhaler, to facilitate processing of the formulation,to provide advantageous powder properties to the formulation, tofacilitate dispersion of the powder from the inhalation device, tostabilize the formulation (for example, antioxidants or buffers), toprovide taste to the formulation, or the like. Advantageously, theadditive does not adversely affect the patient's airways. The insulinderivative can be mixed with an additive at a molecular level or thesolid formulation can include particles of the insulin conjugate mixedwith or coated on particles of the additive. Typical additives includemono-, di-, and polysaccharides; sugar alcohols and other polyols, suchas, for example, lactose, glucose, raffinose, melezitose, lactitol,maltitol, trehalose, sucrose, mannitol, starch, or combinations thereof;surfactants, such as sorbitols, diphosphatidyl choline, or lecithin; orthe like. Typically an additive, such as a bulking agent, is present inan amount effective for a purpose described above, often at about 50% toabout 90% by weight of the formulation. Additional agents known in theart for formulation of a protein such as insulin analogue protein canalso be included in the formulation.

A spray including the insulin derivatives of this invention can beproduced by forcing a suspension or solution of insulin conjugatethrough a nozzle under pressure. The nozzle size and configuration, theapplied pressure, and the liquid feed rate can be chosen to achieve thedesired output and particle size. An electrospray can be produced, forexample, by an electric field in connection with a capillary or nozzlefeed. Advantageously, particles of insulin conjugate delivered by asprayer have a particle size less than about 10 μm, for example in therange of about 1 μm to about 5 μm.

Formulations of insulin derivatives of this invention suitable for usewith a sprayer will typically include the insulin derivative in anaqueous solution at a concentration of about 1 mg to about 20 mg ofinsulin conjugate per ml of solution. The formulation can include agentssuch as an excipient, a buffer, an isotonicity agent, a preservative, asurfactant, and, for example zinc. The formulation can also include anexcipient or agent for stabilization of the insulin derivative, such asa buffer, a reducing agent, a bulk protein, or a carbohydrate. Bulkproteins useful in formulating insulin conjugates include albumin,protamine, or the like. Typical carbohydrates useful in formulatinginsulin conjugates include sucrose, mannitol, lactose, trehalose,glucose, or the like. The insulin derivative formulation can alsoinclude a surfactant, which can reduce or prevent surface-inducedaggregation of the insulin conjugate caused by atomization of thesolution in forming an aerosol. Various conventional surfactants can beemployed, such as polyoxyethylene fatty acid esters and alcohols, andpolyoxyethylene sorbitol fatty acid esters. Amounts will generally rangebetween about 0.001 and about 4% by weight of the formulation.

Pharmaceutical compositions containing an insulin derivative accordingto the present invention may also be administered parenterally topatients in need of such a treatment. Parenteral administration may beperformed by subcutaneous, intramuscular or intravenous injection bymeans of a syringe, optionally a pen-like syringe. Alternatively,parenteral administration can be performed by means of an infusion pump.Further options are to administer the insulin nasally or pulmonally, forexample in compositions, powders or liquids, specifically designed forthe purpose.

Injectable compositions of the insulin derivatives of the invention canbe prepared using the conventional techniques of the pharmaceuticalindustry which involve dissolving and mixing the ingredients asappropriate to give the desired end product. Thus, according to oneprocedure, an insulin derivative according to the invention is dissolvedin an amount of water which is somewhat less than the final volume ofthe composition to be prepared. An isotonic agent, a preservative and abuffer is added as required and the pH value of the solution isadjusted—if necessary—using an acid, e.g. hydrochloric acid, or a base,e.g. aqueous sodium hydroxide as needed. Finally, the volume of thesolution is adjusted with water to give the desired concentration of theingredients.

In a further aspect of the invention the buffer is selected from thegroup consisting of sodium acetate, sodium carbonate, citrate,glycylglycine, histidine, glycine, lysine, arginine, sodium dihydrogenphosphate, disodium hydrogen phosphate, sodium phosphate, andtris(hydroxymethyl)-aminomethan, bicine, tricine, malic acid, succinate,maleic acid, fumaric acid, tartaric acid, aspartic acid or mixturesthereof. Each one of these specific buffers constitutes an alternativeaspect of the invention.

In a further aspect of the invention the formulation further comprises apharmaceutically acceptable preservative which may be selected from thegroup consisting of phenol, o-cresol, m-cresol, p-cresol, methylp-hydroxybenzoate, propyl p-hydroxybenzoate, 2-phenoxyethanol, butylp-hydroxybenzoate, 2-phenylethanol, benzyl alcohol, chlorobutanol, andthiomerosal, bronopol, benzoic acid, imidurea, chlorohexidine, sodiumdehydroacetate, chlorocresol, ethyl p-hydroxybenzoate, benzethoniumchloride, chlorphenesine(3p-chlorphenoxypropane-1,2-diol) or mixturesthereof. In a further aspect of the invention the preservative ispresent in a concentration from 0.1 mg/ml to 20 mg/ml. In a furtheraspect of the invention the preservative is present in a concentrationfrom 0.1 mg/ml to 5 mg/ml. In a further aspect of the invention thepreservative is present in a concentration from 5 mg/ml to 10 mg/ml. Ina further aspect of the invention the preservative is present in aconcentration from 10 mg/ml to 20 mg/ml. Each one of these specificpreservatives constitutes an alternative aspect of the invention. Theuse of a preservative in pharmaceutical compositions is well-known tothe skilled person. For convenience reference is made to Remington: TheScience and Practice of Pharmacy, 19^(th) edition, 1995.

In a further aspect of the invention the formulation further comprisesan isotonic agent which may be selected from the group consisting of asalt (e.g. sodium chloride), a sugar or sugar alcohol, an amino acid(e.g. glycine, L-histidine, arginine, lysine, isoleucine, aspartic acid,tryptophan, threonine), an alditol (e.g. glycerol (glycerine),1,2-propanediol (propyleneglycol), 1,3-propanediol, 1,3-butanediol)polyethyleneglycol (e.g. PEG400), or mixtures thereof. Any sugar such asmono-, di-, or polysaccharides, or water-soluble glucans, including forexample fructose, glucose, mannose, sorbose, xylose, maltose, lactose,sucrose, trehalose, dextran, pullulan, dextrin, cyclodextrin, solublestarch, hydroxyethyl starch and carboxymethylcellulose-Na may be used.In one aspect the sugar additive is sucrose. Sugar alcohol is defined asa C4-C8 hydrocarbon having at least one—OH group and includes, forexample, mannitol, sorbitol, inositol, galactitol, dulcitol, xylitol,and arabitol. In one aspect the sugar alcohol additive is mannitol. Thesugars or sugar alcohols mentioned above may be used individually or incombination. There is no fixed limit to the amount used, as long as thesugar or sugar alcohol is soluble in the liquid preparation and does notadversely effect the stabilizing effects achieved using the methods ofthe invention. In one aspect, the sugar or sugar alcohol concentrationis between about 1 mg/ml and about 150 mg/ml. In a further aspect of theinvention the isotonic agent is present in a concentration from 1 mg/mlto 50 mg/ml. In a further aspect of the invention the isotonic agent ispresent in a concentration from 1 mg/ml to 7 mg/ml. In a further aspectof the invention the isotonic agent is present in a concentration from 8mg/ml to 24 mg/ml. In a further aspect of the invention the isotonicagent is present in a concentration from 25 mg/ml to 50 mg/ml. Each oneof these specific isotonic agents constitutes an alternative aspect ofthe invention. The use of an isotonic agent in pharmaceuticalcompositions is well-known to the skilled person. For conveniencereference is made to Remington: The Science and Practice of Pharmacy,19^(th) edition, 1995.

Typical isotonic agents are sodium chloride, mannitol, dimethyl sulfoneand glycerol and typical preservatives are phenol, m-cresol, methylp-hydroxybenzoate and benzyl alcohol.

Examples of suitable buffers are sodium acetate, glycylglycine, HEPES(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) and sodiumphosphate.

A composition for nasal administration of an insulin derivativeaccording to the present invention may, for example, be prepared asdescribed in European Patent No. 272097 (to Novo Nordisk A/S).

Compositions containing insulin derivatives of this invention can beused in the treatment of states which are sensitive to insulin. Thus,they can be used in the treatment of type 1 diabetes, type 2 diabetesand hyperglycaemia for example as sometimes seen in seriously injuredpersons and persons who have undergone major surgery. The optimal doselevel for any patient will depend on a variety of factors including theefficacy of the specific insulin derivative employed, the age, bodyweight, physical activity, and diet of the patient, on a possiblecombination with other drugs, and on the severity of the state to betreated. It is recommended that the daily dosage of the insulinderivative of this invention be determined for each individual patientby those skilled in the art in a similar way as for known insulincompositions.

Where expedient, the insulin derivatives of this invention may be usedin mixture with other types of insulin, e.g. insulin analogues with amore rapid onset of action. Examples of such insulin analogues aredescribed e.g. in the European patent applications having thepublication Nos. EP 214826 (Novo Nordisk A/S), EP 375437 (Novo NordiskA/S) and EP 383472 (Eli Lilly & Co.).

The present invention is further illustrated by the following exampleswhich, however, are not to be construed as limiting the scope ofprotection.

DESCRIPTION OF DRAWINGS

FIG. 1: Size-exclusion chromatography of the insulin derivative inexample 2 in mixture with insulin aspart (B28Asp human insulin). Insulincontent in each individual peak is quantified by HLPC. The insulinderivative and Aspart in formulations with either 2.1 Zn(II) per hexameror 6 Zn(II) per hexamer elutes as two separate fractions (high-molecularweight insulin and medium molecular weight insulin, respectively). TheSEC experiments are performed in accordance with example 20.

FIG. 2: Clamp action profile after subcutaneous injection of the insulinderivative described in example 2 in different concentrations and withdifferent Zn(II) concentrations demonstrating that the action profile ofthe insulin derivative is similar whether the insulin derivative isadministered with 2.3 or 6 Zn(II) per six insulin or as 600 μM or 1200μM formulations. The clamp experiment is performed in accordance withexample 21.

FIG. 3: Clamp action profile after subcutaneous injection of the insulinderivative described in example 2 in mixture with insulin Aspart, or asindividual injections, demonstrating that there is no significantblunting of the individual insulin action profiles. The clamp experimentis performed in accordance with example 21.

FIG. 4: Clamp action profile after subcutaneous injection of the insulinderivative in example 2 in three doses demonstrating long effect ofinsulin action. The clamp experiment is performed in accordance withexample 21.

EXAMPLES Example 1 Synthesis ofN^(εB29)-ω-carboxy-pentadecanoyl-γ-L-glutamylamide desB30 human insulin

Step 1: Mono-tert-butyl hexadecandioate

Hexadecadioic acid (40.0 g, 140 mmol) was suspended in toluene (250 ml)and the mixture was heated to reflux. N,N-dimethylformamidedi-tert-butyl acetal (76.3 g, 375 mmol) was added drop-wise over 4hours. The mixture was refluxed overnight. The solvent was removed invacuo at 50° C., and the crude material was suspended in DCM/AcOEt (500ml, 1:1) and stirred for 15 mins. The solids were collected byfiltration and triturated with DCM (200 ml). The filtrated wereevaporated in vacuo to give crude mono-tert-butyl hexadecandioate, 30grams. This material was suspended in DCM (50 ml), cooled with ice for10 mins, and filtered. The solvent was removed in vacuo to leave 25 gramcrude mono-tert-butyl hexadecandioate, which was recrystallized fromheptane (200 ml) to give mono-tert-butyl hexadecandioate, 15.9 g (33%).

¹H-NMR (CDCl₃) δ: 2.35 (t, 2H), 2.20 (t, 2H), 1.65-1.55 (m, 4H), 1.44(s, 9H), 1.34-1.20 (m, 20H).

Step 2: Succinimidyl tert-butyl hexadecandioate

The mono tert-butyl ester (2 g, 5.8 mmol) was dissolved in THF (20 ml)and treated with TSTU (2.1 g, 7.0 mmol) and DIEA (1.2 ml, 7.0 mmol) andstirred overnight. The mixture was filtered, and the filtrate wasevaporated in vacuo. The residue was dissolved in AcOEt and washed twicewith cold 0.1 M HCl and water. Drying over MgSO₄ and evaporation invacuo gave succinimidyl tert-butyl hexadecandioate, 2.02 g (79%).

¹H-NMR (CDCl₃) δ: 2.84 (s, 4H), 2.60 (t, 2H), 2.20 (t, 2H), 1.74 (p,2H), 1.56 (m, 2H), 1.44 (s, 9H), 1.40 (m, 2H), 1.30-1.20 (m, 18H).

Step 3: ω-tert-butyl-carboxy-pentadecanoyl-L-glutamylamide

Succinimidyl tert-butyl hexadecandioate (100 mg, 0.227 mmol) wasdissolved in DMF (2 ml) and treated with L-glutamylamide (37 mg, 0.25mmol) and DIEA (58 μl, 0.34 mmol) and the mixture was stirred overnight.The solvent was evaporated in vacuo, and the crude product was dissolvedin AcOEt, and washed twice with 0.2M HCl, with water and brine. Dryingover MgSO₄ and evaporation in vacuo gaveω-tert-butyl-carboxy-pentadecanoyl-L-glutamyl amide, 85 mg (80%).

¹H-NMR (CDCl₃) δ: 6.98 (s, 1H), 6.60 (d, 1H), 5.88 (s, 1H), 4.69 (m,1H), 2.55-2.41 (m, 2H), 2.25-2.18 (m, 2H), 2.14 (m, 1H), 1.93 (m, 1H),1.65-1.54 (m, 4H) 1.44 (s, 9H), 1.27 (br, 20H).

Step 4: ω-tert-butyl-carboxy-pentadecanoyl-L-glutamylamideγ-succinimidyl ester

ω-tert-butyl-carboxy-pentadecanoyl-L-glutamylamide (85 g, 0.181 mmol)was dissolved in THF (1 ml) and treated with TSTU (65 g, 0.217 mmol) andDIEA (37 μl, 0.217 mmol) and stirred overnight. The mixture wasfiltered, and the filtrate was evaporated in vacuo. The residue wasdissolved in AcOEt and washed twice with cold 0.1 M HCl and water.Drying over MgSO₄ and evaporation in vacuo gaveω-tert-butyl-carboxy-pentadecanoyl-L-glutamyl amide γ-succinimidylester, 91 mg (89%).

¹H-NMR (CDCl₃) δ: 6.59 (s, 1H), 6.41 (d, 1H), 5.56 (s, 1H), 4.62 (m,1H), 3.02-2.94 (dd, 2H), 2.84 (s, 4H), 2.71-2.58 (m, 2H), 1.76 (m, 1H),1.53-1.63 (m, 4H), 1.44 (s, 9H), 1.25 (br, 20H).

Step 5: N^(εB29)-ω-carboxy-pentadecanoyl-γ-L-glutamylamide desB30 humaninsulin

DesB30 human insulin (500 mg, 0.088 mmol) was dissolved in 100 mM Na₂CO₃(5 ml, pH 10.2) at room temperature.ω-Tert-butyl-carboxy-pentadecanoyl-L-glutamyl amide γ-succinimidyl ester(57 mg, 0.105 mmol), was dissolved in acetonitrile (5 ml) andsubsequently added to the insulin solution. After 30 mins, 0.2 Mmethylamine (0.5 ml) was added. pH was adjusted by HCl to 5.5, and theisoelectric precipitate was collected by centrifugation and dried invacuo to give 423 mg. The coupling yield was 42% (RP-HPLC, C4 column;Buffer A: 10% MeCN in 0.1% TFA-water, Buffer B: 80% MeCN in 0.1%TFA-water; gradient 20% to 90% B in 16 minutes). The protected productwas dissolved in 95% TFA (12 ml), left 30 mins, and evaporated in vacuo.The crude product was dissolved in water and lyophilized.

N^(εB29)-ω-carboxy-pentadecanoyl-γ-L-glutamylamide desB30 human insulinwas purified by RP-HPLC on C4-column, buffer A: 20% EtOH+0.1% TFA,buffer B: 80% EtOH+0.1% TFA; gradient 15-60% B, followed by HPLC onC4-column, buffer A: 10 mM Tris+15 mM ammonium sulphate in 20% EtOH, pH7.3, buffer B: 80% EtOH, gradient 15-60% B. The collected fractions weredesalted on Sep-Pak with 70% acetonitrile+0.1% TFA, neutralized byaddition of ammonia and freeze-dried. The unoptimized yield was 50 mg,12%. The purity as evaluated by HPLC was >98%. LCMS 6102.8;C₂₇₄H₄₁₂N₆₆O₈₀S₆ requires 6103.1.

Example 2 Synthesis of N^(εB29)-ω-carboxy-pentadecanoyl-γ-amino-butanoyldesB30 human insulin

This compound was prepared from hexadecandioic acid and γ-aminobutyricacid, in analogy with example 1.

ω-tert-butyl-carboxy-pentadecanoyl-γ-amino-butyric acid succinimidylester

¹H-NMR (CDCl₃) δ: 5.80 (m, 1H), 3.36 (dd, 2H), 2.84 (s, 4H), 2.65 (t,2H), 2.21-2.13 (m, 4H), 1.99 (p, 2H), 1.44 (s, 9H), 1.66-1.51 (m, 6H),1.25 (br, 20H).

N^(εB29)-ω-carboxy-pentadecanoyl-γ-amino-butanoyl desB30 human insulin

LCMS 6059.9; C₂₇₃H₄₁₁N₆₅O₇₉S₆ requires 6060.1.

Example 3 Synthesis ofN^(εB29)-ω-carboxy-tetradecanoyl-γ-L-glutamylamide desB30 human insulin

This compound was prepared from pentadecandioic acid and L-glutamylamidein analogy with example 1.

LCMS 6088.2; C₂₇₃H₄₁₀N₆₆O₈₀S₆ requires 6089.1.

Example 4 Synthesis of N^(εB29)-ω-carboxy-tridecanoyl-γ-L-glutamylamidedesB30 human insulin

This compound was prepared from tetradecandioic acid and L-glutamylamidein analogy with example 1.

LCMS 6075.3; C₂₇₂H₄₀₈N₆₆O₈₀S₆ requires 6075.1.

Example 5 Synthesis of N^(εB29)-ω-carboxy-pentadecanoyl-β-alanyl desB30human insulin

This compound was prepared from hexadecandioic acid and β-alanine inanalogy with example 1.

LCMS 6044.8; C₂₇₂H₄₀₉N₆₅O₇₉S₆ requires 6046.1.

Example 6 Synthesis ofN^(εB29)-ω-carboxy-pentadecanoyl-γ-L-aspartylamide desB30 human insulin

This compound was prepared from hexadecandioic acid and L-aspartylamidein analogy with example 1.

LCMS 6088.8; C₂₇₃H₄₁₀N₆₆O₈₀S₆ requires 6089.1.

Example 7 Synthesis of N^(εB29)-ω-carboxy-pentadecanoyl-ε-aminohexanoyldesB30 human insulin

This compound was prepared from hexadecandioic acid and ε-amino-hexanoicacid in analogy with example 1.

LCMS 6086.1; C₂₇₅H₄₁₅N₆₅O₇₉S₆ requires 6088.1.

Example 8 Synthesis of N^(εB29)-ω-carboxy-pentadecanoyl-δ-aminopentanoyldesB30 human insulin

This compound was prepared from hexadecandioic acid andδ-amino-pentanoic acid in analogy with example 1.

LCMS 6074.2, C₂₇₄H₄₁₃N₆₅O₇₉S₆ requires 6074.1.

Example 9 Synthesis ofN^(εB29)-10-(4-carboxyphenoxy)-decanoyl-γ-L-glutamylamide desB30 humaninsulin

Step 1: 4-Hydroxy-benzoic acid tert-butyl ester

4-Hydroxy-benzoic acid (3 g, 21.7 mmol) was stirred in toluene (35 ml,dried over mol. sieves). The solution was heated to 80° C. under N₂, andN,N′-dimethylformamide di-tert-butyl acetal (10.42 mL, 43.4 mmol) wasadded over ca. 5 min. The mixture was stirred at 80° C. for 1 h 10 min.,and cooled to rt. The solution was washed with water, twice with sat.NaHCO₃ and sat. NaCl (15 mL each), dried over MgSO₄, and concentrated toyield a yellow oil (2.77 g). The product was purified by flashchromatography (380 g silica, eluant: 4:6 AcOEt/heptane (2 L) and 1:1AcOEt/heptane 700 mL) to yield white crystals (2.07 g, 49% yield).

HPLC-MS m/z: 217 (M+23).

¹H-NMR (CDCl₃, 400 MHz) 7.90 (d, 2H), 6.85 (d, 2H), 6.10 (s, 1H), 1.59(s, 9H).

Step 2: 4-(9-Methoxycarbonylnonyloxy)benzoic acid tert-butyl ester

4-Hydroxy-benzoic acid tert-butyl ester (500 mg, 2.57 mmol) and10-bromodecanoic acid methyl ester (683 mg, 2.57 mmol) were dissolved inacetonitrile, and K₂CO₃ (712 mg, 5.15 mmol) was added. The mixture wasrefluxed under nitrogen for 16 h, and cooled to rt. The solids werefiltered off, and the filtrate concentrated under vacuum. The resultingresidue was dissolved in AcOEt (50 ml) and water (25 mL). The phaseswere separated and the organic phase was dried over MgSO₄ andconcentrated to yield a colorless oil (874 mg, 90% yield).

HPLC-MS m/z: 402 (M+23).

¹H-NMR (CDCl₃, 400 MHz) 7.92 (d, 2H), 6.87 (d, 2H), 3.99 (t, 2H), 3.67(s, 3H), 2.31 (t, 2H), 1.72-1.83 (m, 2H), 1.59-1.69 (m, 2H), 1.58 (s,9H), 1.40-1.50 (m, 2H), 1.23-1.40 (br, 8H).

Step 3: 4-(9-Carboxynonyloxy)benzoic acid tert-butyl ester

4-(9-Methoxycarbonylnonyloxy)benzoic acid tert-butyl ester (858 mg, 2.27mmol) was dissolved in THF (5 ml), and 1N NaOH (2.27 mmol) was added.The mixture was stirred for 16 h. AcOEt (40 mL) and 1N HCl (2.38 ml) inwater (25 ml) were added. The phases were separated, and the organicphase was dried over MgSO₄, and concentrated under vacuum to yield awhite solid (781 mg, 95% yield).

HPLC-MS m/z: 387 (M+23).

¹H-NMR (CDCl₃, 400 MHz) 7.92 (d, 2H), 6.87 (d, 2H), 3.99 (t, 2H), 2.35(t, 2H), 1.73-1.84 (m, 2H), 1.60-1.69 (m, 2H), 1.58 (s, 9H), 1.39-1.51(m, 2H), 1.24-1.39 (br, 8H).

Step 4: 4-[9-(2,5-Dioxopyrrolidin-1-yloxycarbonyl)nonyloxy]benzoic acidtert-butyl ester

4-(9-Carboxynonyloxy)benzoic acid tert-butyl ester (779 mg, 2.14 mmol)was dissolved in THF (15 mL), and DIEA (366 μl, 2.14 mmol) was added.The mixture was cooled to 0° C., and placed under nitrogen, and HSTU wasadded. The mixture was stirred at 0° C. for 30 min and at RT for 16 h.The sample was concentrated under vacuum and AcOEt (40 ml) was added.The mixture was washed with 0.2 N HCl (2×25 ml), dried over MgSO₄, andconcentrated under vacuum to yield a slightly yellow solid. The solidwas recrystallized from AcOEt to yield a white powder (276 mg, 28%yield). The mother liquor was concentrated to yield crystalline residue(430 mg, 43% yield). Data for the white powder:

HPLC-MS m/z: 484 (M+23).

¹H-NMR (CDCl₃, 300 MHz) 7.93 (d, 2H), 6.88 (d, 2H), 3.99 (t, 2H), 2.83(s, 4H), 2.61 (t, 2H), 1.67-1.88 (m, 4H), 1.58 (s, 11H, theoret. 9H+water), 1.27-1.52 (m, 10H).

Step 5: 4-[9-((S)-1-Carbamoyl-3-carboxypropylcarbamoyl)nonyloxy]benzoicacid tert-butyl ester

4-[9-(2,5-Dioxopyrrolidin-1-yloxycarbonyl)nonyloxy]benzoic acidtert-butyl ester (200 mg, 0.433 mmol) was stirred in DMF (2 mL) andH-Glu-NH₂ (63 mg) was added. The non-homogeneous mixture was stirred atrt for 16 h. LC/MS analysis indicated the reaction had not gone tocompletion. H-Glu-NH₂ (20 mg) and more DMF (2 mL) were added and themixture was stirred for 2 d at rt. The sample was concentrated undervacuum and AcOEt (50 mL) was added. The solution was washed with 0.2 NHCl (2×25 mL) and water (25 mL), dried over MgSO₄, and concentratedunder vacuum to yield a white solid (180 mg, 86% yield).

HPLC-MS m/z: 493 (M+1).

Step 6:4-{9-[(S)-1-Carbamoyl-3-(2,5-dioxopyrrolidin-1-yloxycarbonyl)propylcarbamoyl]nonyloxy}benzoicacid tert-butyl ester

The HSTU activation was performed in manner similar to that describedfor 4-[9-(2,5-Dioxopyrrolidin-1-yloxycarbonyl)nonyloxy]benzoic acidtert-butyl ester. The product was purified by flash chromatography (1:1AcOEt:heptane and AcOEt) to yield 18 mg.

HPLC-MS m/z: 590 (M+1).

Step 7: N^(εB29)-10-(4-carboxyphenoxy)decanoyl-γ-L-glutamylamide desB30insulin

DesB30 insulin (126 mg, 0.022 mmol) was dissolved by adding 100 mMNa₂CO₃ (1.5 mL) and acetonitrile (1.5 mL) in a 10 ml round bottom-flask.4-{9-[(S)-1-Carbamoyl-3-(2,5-dioxopyrrolidin-1-yloxycarbonyl)propylcarbamoyl]nonyloxy}benzoicacid tert-butyl ester (14 mg, 0.022 mmol) was added in acetonitrile (750uL) and Na₂CO₃ (750 uL) was added so the final solution was 50:50 100 mMNa₂CO₃/acetonitrile. The solution was stirred at RT for 1 h. Thesolution was transferred to a 15 ml centrifuge tube, washing withMilli-Q water (6 ml). The solution was cooled on ice, and the pH wasadjusted to 5.1 by adding 1N HCl, which lead to precipitation. The tubewas centrifuged at 5000 rpm for 10 min at 10° C. The solvent wasdecanted from the solid. 95:5 TFA/water (2.5 ml) was added to the solid.The solution was poured into a round bottom flask, washing with more95:5 TFA/water (2.5 ml). The solution was stirred for 30 min at RT, andconcentrated under vacuum. DCM was added and removed twice, and theflask was placed under vacuum at RT. The product was purified bypreparative HPLC (C18 column, acetonitrile/water/0.05% TFA). Therelevant fractions were pooled (two batches) and diluted 1:1 with water.The solutions were cooled on ice, and the precipitation was induced byadjusting the pH to ca. 5 with 1 N NaOH. The samples were centrifuged(5000 rpm, 10 min, 5° C.). The liquid was decanted off and the pelletswere lyophilized to yield a white solid (22 mg+12 mg).

MALDI-MS (alpha-cyano-4-hydroxycinnamic acid) m/z: 6128.7 (M=6125.1).

HPLC-MS m/z: 1532.8 ((M+4)/4=1532.2).

Example 10 Synthesis of N^(εB29)-4-[11-(4-Carboxyphenyl)undecanoylamino]butyryl desB30 human insulin

Step 1: 4-Iodobenzoic acid tert-butyl ester

4-Iodobenzoic acid (10 g, 40.3 mmol) was dissolved in dry toluene (100ml, dried over mol. sieves). The solution was heated to 70° C. under aflow of nitrogen. A solution of N,N′-dimethylformamide di-tert-butylacetal (24.6 g, 121 mmol) in toluene (25 mL) was added over ca. 30 min.The reaction was mixed for 16 h. At some point the heating unit failed,so the reaction cooled from 70° C. to rt. The solution was heated to 70°C. for and mixed for 5 h. The sample was concentrated under vacuum, andAcOEt (400 ml) was added. The solution was then washed with 1:1 sat.NaHCO₃/water (150 ml), and sat. NaHCO₃, water and sat. NaCl (75 mLeach). The organic phase was dried (MgSO₄) and concentrated under vacuumto yield light brown oil.

HPLC-MS m/z: 327 (M+23).

¹H-NMR (CDCl₃, 400 MHz) δ 7.77 (d, 2H), 7.69 (d, 2H), 1.58 (s, 9H).

Step 2: 11-Iodo undecanoic acid methyl ester

11-Bromo undecanoic acid methyl ester (20.2 g, 72.3 mmol) was dissolvedin acetone (200 ml). Sodium iodide (54 g, 361 mmol) was added andreaction was refluxed under nitrogen for 16 h. After cooling to RT thesalts were filtered off. The filtrate was concentrated under vacuum andwater (200 ml) was added. The solution was extracted with AcOEt (2×100ml) adding some sat. NaCl to aid phase separation. The organic extractswere pooled and washed with water (100 ml) plus a little sat. NaCl, andsat. NaCl (50 mL). Dry over MgSO₄. The solution was a red-orange color.Three teaspoons of activated charcoal were added. After mixing, thesolution was filtered through a bed of celite. The filtrate wasconcentrated under vacuum to yield a light yellow oil (20.96 g, 89%).

HPLC-MS m/z: 327 (M+1).

¹H-NMR (CDCl₃, 300 MHz) δ 3.67 (s, 3H), 3.19 (t, 2H), 2.30 (t, 2H),1.74-1.89 (m, 2H), 1.53-1.70 (m, 2H), 1.34-1.46 (m, 2H), 1.28 (br, 10H).

Step 3: 4-(10-Methoxycarbonyldecyl)benzoic acid tert-butyl ester

All glassware was dried prior to use. THF was dried over molecularsieves. LiCl was dried at 150° C. for 1 h, then stored in a closedbottle. All reaction solutions were made under nitrogen, and thesolutions were transferred via syringe. 4-Iodobenzoic acid tert-butylester (1.2 g, 3.95 mmol) was dissolved in THF (3 ml) and cooled to −30°C. Isopropyl magnesium chloride (4.34 mmol, 2M in THF) was added over 5minutes, and the solution was stirred for 1 hr at a temperature between−18° C. to −25° C. The solution was cooled to −22° C., and a mixture ofCuCN (0.389 g, 4.34 mmol) and LiCl (0.368 g, 8.68 mmol) in THF (4.2 ml)was then added. The reaction vessel was removed from cooling and allowedto warm to RT (ca. 10 min). Trimethylphosphite (0.95 mL) was added, andafter stirring for 5 min at rt, a solution of 11-iodo-undecanoic acidmethyl ester (1.0 g, 3.16 mmol) in THF (3 ml) was added. The solutionwas mixed at rt for 16 h. Sat. NH₄Cl (3 ml) was added, and the solutionwas poured into water (60 mL). The solution was extracted with AcOEt(3×35 ml). The organic extracts were pooled and washed with water (30mL) using some Sat. NaCl to aid phase separation. The solvent wasremoved under vacuum to yield a biphasic residue. AcOEt (ca 2 ml) wasadded and the flask was swirled gently. Not all of the thick whiteresidue dissolved. The portion which dissolved was added to a column ofsilica (50 g) and eluted with AcOEt: heptane 1:11. The appropriatefractions were concentration under vacuum to yield an oil (1.25 g). Theoil was dissolved in acetone (30 mL), and piperidine (1 mL) was added.NaI (0.8 g) was added and the mixture was stirred and refluxed for 16 h.The mixture was concentrated under vacuum and partitioned between AcOEt(50 mL) and 1 N HCl (25 mL). The organic phase was washed with 1 N HCl(2×25 mL), dried over MgSO₄, and concentrated under vacuum to yield acolorless oil (1.1 g). The product was purified by flash chromatography(eluant: AcOEt:heptane 1:11, 150 g silica) to yield a colorless oil(0.72 g, 61%).

HPLC-MS m/z: 399 (M+23).

¹H-NMR (CDCl₃, 300 MHz) δ 7.90 (d, 2H), 7.21 (d, 2H), 3.66 (s, 3H), 2.64(t, 2H), 2.30 (t, 2H), 1.48-1.70 (m, 13H), 1.27 (br, 12H).

Step 4: 4-(10-Carboxydecyl)benzoic acid tert-butyl ester

The compound was prepared in analogous fashion to the procedure used inthe preparation of 4-(9-Carboxynonyloxy)benzoic acid tert-butyl ester toyield a white solid (0.68 g).

HPLC-MS m/z: 385 (M+23).

¹H-NMR (CDCl₃, 300 MHz) δ 7.90 (d, 2H), 7.21 (d, 2H), 2.64 (t, 2H), 2.34(t, 2H), 1.53-1.71 (m, 13H), 1.28 (br, 12H).

Step 5: 4-[10-(2,5-Dioxopyrrolidin-1-yloxycarbonyl)decyl]benzoic acidtert-butyl ester

The compound was prepared in analogous fashion to the procedure used inthe preparation of4-[9-(2,5-Dioxopyrrolidin-1-yloxycarbonyl)nonyloxy]benzoic acidtert-butyl ester.

HPLC-MS m/z: 482 (M+23).

¹H-NMR (CDCl₃, 400 MHz) δ 7.89 (d, 2H), 7.21 (d, 2H), 2.76-2.93 (m, 4H),2.54-2.68 (m, 2H), 1.67-1.81 (M, 2H), 1.52-1.66 (m, 11H), 1.35-1.43 (M,2H), 1.19-1.35 (br, 10H).

Step 6: 4-[10-(3-Carboxy-propylcarbamoyl)decyl]benzoic acid tert-butylester

4-[10-(2,5-Dioxopyrrolidin-1-yloxycarbonyl)decyl]benzoic acid tert-butylester (300 mg, 0.65 mmol) was dissolved in DMF (3 ml) and 4-aminobutyric acid (67 mg, 0.65 mmol). The mixture was stirred for 16 h undernitrogen. The solvent was removed under vacuum and AcOEt (35 ml) wasadded. The solution was washed with 0.2 N HCl and water (15 ml each).Sat. NaHCO₃ was added (not intended) to the organic phase. DCM (50 ml)was added. Some of the organic phase was removed and DCM (100 ml) wasadded to the aqueous phase and allowed to stand overnight. The mixturewas cooled on ice and the pH was adjusted to 1.9 with 4N HCl. Theorganic phase was isolated, dried over MgSO₄ and concentrated undervacuum to yield on oil (220 mg, 76% yield).

HPLC-MS m/z: 470 (M+23).

¹H-NMR (CDCl₃, 400 MHz) δ 7.89 (d, 2H), 7.21 (d, 2H), 5.79 (br, 1H),3.27-3.40 (m, 2H), 2.64 (t, 2H), 2.40 (t, 2H), 2.18 (t, 2H), 1.78-1.91(m, 2H), 1.51-1.61 (m, 13H), 1.35-1.43 (M, 2H), 1.17-1.36 (br, 12H).

Step 7:4-{10-[3-(2,5-Dioxo-pyrrolidin-1-yloxycarbonyl)propylcarbamoyl]decyl}benzoicacid tert-butyl ester

The compound was prepared in analogous fashion to the procedure used inthe preparation of4-[9-(2,5-Dioxopyrrolidin-1-yloxycarbonyl)nonyloxy]benzoic acidtert-butyl ester, but TSTU was used instead of HSTU. Precipitation(DCM/Heptane) yielded white crystals (180 mg, 70% yield).

HPLC-MS m/z: 568 (M+23).

¹H-NMR (CDCl₃, 400 MHz) δ 7.89 (d, 2H), 7.21 (d, 2H), 5.83 (br, 1H),3.30-3.43 (m, 2H), 2.85 (br, 4H), 2.57-2.73 (m, 4H), 2.15 (t, 2H),1.92-2.07 (m, 2H), 1.56-1.64 (m, 13H), 1.18-1.36 (br, 12H).

Step 8: N^(εB29)-4-[11-(4-carboxyphenyl)undecanoylamino]butyryl desB30human insulin

The compound was prepared in analogous fashion to the procedure used inthe preparation of example 9 to yield 30 mg.

MALDI-MS (alpha-cyano-4-hydroxycinnamic acid) m/z: 6067 (M=6080,reference standard (M=5706) showed M−13).

HPLC-MS m/z: 1520.9 ((M+4)/4=1521).

Example 11 Synthesis ofN^(εB29)-(3-(3-{4-[3-(7-carboxyheptanoylamino)propoxy]butoxy}propylcarbamoyl)-propionyl-γ-glutamylamide)desB30 human Insulin

Step 1:N-{3-[4-(3-tert-Butoxycarbonylaminopropoxy)-butoxy]-propyl}succinamicacid

123-0000-3007

1-(tert-Butoxycarbonylamino)-4,9-dioxa-12-dodecanamine (5.0 g, 16.45mmol) was dissolved in THF (30 mL), succinic anhydride (1.81 g, 18.1mmol) in acetonitrile (10 mL) was added and the mixture was heated to 60C for 4 h, and subsequently stirred at RT overnight.

The mixture was evaporated to dryness and EtAc (50 mL) was added.

The EtAc phase was washed with HCl (0.1 M) 3 times, dried with MgSO₄ andsubsequently the organic phase was evaporated to dryness which gave 5.86g (88%) of thick oil.

LCMS: Rt 2.86 min; m/z (M+1) 405. Calcd: 405.

This product was used without further purification.

Step 2: Octanedioic acid tert-butyl ester 2,5-dioxo-pyrrolidin-1-ylester

Octanedioic acid mono-tert-butyl ester (3.14 g, 13.63 mmol) wasdissolved in THF (100 mL). TSTU (4.9 g, 16.3 mmol) was added and pH wasadjusted to 8.5 with DIPEA (2.85 mL).

The mixture was stirred under nitrogen overnight, evaporated to dryness,dissolved in EtAc (50 mL) which subsequently was extracted 2 times withHCL (0.1 M). The organic phase was dried with MgSO₄, filtered andevaporated resulting in an slightly yellow oil (5 g, containing smallamounts of solvent).

LCMS: Rt 6.56 min; m/z (M+1) 328. Calcd: 328.

Step 3:7-(3-{4-[3-(3-carboxypropionylamino)propoxy]butoxy}propylcarbamoyl)heptanoicacid tert-butyl ester

123-0000-3012

N-{3-[4-(3-tert-Butoxycarbonylaminopropoxy)-butoxy]-propyl}succinamicacid (4.60 g, 11.37 mmol) was stirred with TFA (20 mL) at RT for 60 min,after evaporation the residue was stripped with DCM (30 mL×2) andevaporated to dryness.

The resulting oil was dissolved in acetonitrile (30 mL) and octanedioicacid tert-butyl ester 2,5-dioxo-pyrrolidin-1-yl ester (4.46 g, 13.6mmol) in DMF (20 mL) was added.

pH was adjusted to 8.5 with DIPEA and the mixture was stirred overnightunder nitrogen. The mixture was subsequently evaporated to dryness andredissolved in EtOAc (50 mL). The EtOAc phase was extracted ×3 with HCl(0.1 M), the organic layer dried over magnesium sulphate, filtered andevaporated resulting in a slightly yellow crystalline oil (6.5 g,content of solvent residues).

LCMS: Rt 4.31 min; m/z (M+1) 517. Calcd: 517.

The crude product was used for further reaction without furtherpurification.

Step 4:7-[3-(4-{3-[3-((S)-1-carbamoyl-3-carboxypropylcarbamoyl)propionylamino]propoxy}butoxy)propylcarbamoyl]heptanoicacid tert-butyl ester

0123-0000-3078

7-(3-{4-[3-(3-Carboxypropionylamino)propoxy]butoxy}propylcarbamoyl)heptanoicacid tert-butyl ester (2.4 g), the crude product from step 3, wasdissolved in THF (60 mL), TSTU (2.11 g, 6.97 mmol) was added togetherwith DMF (10 mL), pH was adjusted to 8.2 with DIPEA (0.8 mL). Themixture was stirred overnight under nitrogen.

The mixture was evaporated and the residue dissolved in EtOAc which wasextracted with HCl (0.1 M) 3 times. The organic layer was dried withmagnesium sulphate, filtered and the filtrate evaporated to give 3.2 goil.

LCMS: Rt 4.57 min; m/z 614, corresponding to the activated acid.

This crude product was dissolved in acetonitrile (40 mL) and L-glutamicacid amide (0.6 g, 4.1 mmol) was added together with DMF (5 mL), pH wasadjusted to 8.2 with DIPEA (1.4 mL).

The mixture was stirred at RT for overnight; filtration followed byevaporation afforded thick yellow oil.

This was extracted between EtOAc and HCl (0.1 M) as reported above, andthe resulting dried EtOAc layer gave 1.66 g crude product onevaporation.

LCMS: Rt 3.62 min; m/z (M+1) 645. The crude product was purified bypreparative HPLC using acetonitrile/water/0.1% TFA as eluent on C18column (Jones, Kromasil RP18 5 μm 15×225 mm). Gradient: 0.0-10.0 min 35%acetonitrile A; 10.0-30.0 min 35-90% A; The product was collected infractions from 16-18 min. The combined fractions were evaporatedyielding the wanted product (1.0 g). LCMS: Rt 3.59 min; m/z (M+1) 645,calcd. 645.

Step 5: N^(εB29)-(3-(3-{4-[3-(7carboxyheptanoylamino)propoxy]butoxy}propylcarbamoyl)propionyl-γ-L-glutamylamide)desB30 human insulin

7-[3-(4-{3-[3-((S)-1-Carbamoyl-3-carboxypropylcarbamoyl)propionylamino]propoxy}butoxy)-propylcarbamoyl]heptanoicacid tert-butyl ester from step 4 (1.0 g, 1.55 mmol) was dissolved inTHF (20 mL), TSTU (0.51 g, 1.7 mmol) was added and pH adjusted to >8with DIPEA (0.27 mL).

The mixture was stirred overnight under nitrogen. Evaporation followedby extraction between EtOAc and HCl (0.1 M), drying of the organic phase(MgSO₄) followed by evaporation to dryness gave 21 mg oil, LCMS: Rt.4.34 min, m/z 742.

This crude product was dissolved in acetonitrile (10 mL), pH wasadjusted to 8 with Na₂CO₃ (0.1 M) and added to a solution of desB30human insulin (1 g) dissolved in Na₂CO₃ solution (15 mL, pH 10.2).

The mixture was stirred under nitrogen at RT for 1 h. Then pH wasadjusted to 5.4 by means of HCl (2M) resulting in precipitation. Themixture was filtered, the filtrate freeze dried, and the precipitatedried in vacuum overnight.

Both fractions were purified on Gilson using acetonitrile/water/0.1% TFAas eluent on C18 column (Jones, Kromasil RP18 5 μm 15×225 mm).

Gradient: 0.0-5.0 min 35% acetonitrile (A); 5.0-25.0 min 35-90% A; Theproduct was collected in fractions from 12-15 min. The combinedfractions were evaporated, re-dissolved in water and freeze-driedyielding 27 mg of the wanted product.

LCMS: Rt. 7.76 min, m/z 1570

MALDI-MS (sinnapinic acid): 6277; C₂₈₀H₄₂₂N₆₈O₈₄S₆ requires 6277.

Example 12 Synthesis of N^(εB29)-ω-carboxy-tridecanoyl-γ-amino-butanoyldesB30 human insulin

This compound was prepared from tetradecandioic acid and γ-amino-butyricacid in analogy with example 1.

LCMS 6032.1, C₂₇₁H₄₀₇N₆₅O₇₉S₆ requires 6032.0.

Example 13 Synthesis of N^(εB29)-ω-carboxy-undecanoyl-δ-amino-butanoyldesB30 human insulin

This compound was prepared from dodecandioic acid and γ-amino-butyricacid in analogy with example 1.

LCMS 6003.8, C₂₆₉H₄₀₃N₆₅O₇₉S₆ requires 6004.0.

Example 14 Synthesis ofN^(εB29)-ω-carboxy-tetradecanoyl-γ-amino-butanoyl desB30 human insulin

This compound was prepared from pentadecandioic acid and γ-aminobutyricacid in analogy with example 1.

LCMS 6045.6, C₂₇₂H₄₀₉N₆₅O₇₉S₆ requires 6046.1.

Example 15 Synthesis ofN^(εB29)-{4-[10-(4-Carboxy-phenoxy)-decanoylamino]-butyryl} desB30insulin

This compound was prepared from 4-(9-methoxycarbonylnonyloxy)benzoicacid tert-butyl ester in analogy with examples 9 and 10.

LCMS: 6095.6, C₂₇₆H₄₀₉N₆₅O₇₉S₆ requires 6094.1.

Example 16 Synthesis ofN^(εB29)-{4-[(14-Carboxy-tetradecanoylamino)-methyl]-benzoyl} desB30insulin

This compound was prepared from 4-aminomethyl benzoic acid in analogywith example 1.

LCMS: 6082.0, C₂₇₅H₄₀₆N₆₆O₈₁S₆ requires 6082.1.

Example 17 Synthesis of N^(εB29)-[16-(4-Carboxy-phenoxy)-hexadecanoyl]desB30 insulin

Step 1: 16-Bromohexadecanoic acid methyl ester

16-Bromohexadecanoic acid (6 g, 17.9 mmol) was dissolved in methanol (35mL), toluene (100 mL) and trimethylorthoformate (20 mL). Amberlyst 15was added and the mixture was stirred under nitrogen for 16 h at 55° C.The mixture was concentrated, and dissolved in methanol (ca. 50 mL) andDCM (30 mL). The resin was filtered off, and the filtrate wasconcentrated. The volume was increased to ca. 40 mL with methanol.Cooling produced crystals which were filtered off, washed with coldmethanol and dried to yield white crystals (5.61 g, 90% yield).

¹H-NMR (DMSO, 300 MHz) 3.57 (s, 3H), 3.52 (t, 2H), 2.28 (t, 2H), 1.78(m, 2H), 1.50 (m, 2H), 1.37 (m, 2H).

The remainder of the steps were performed in analogy with example 9.

LCMS: 6081.2, C₂₇₆H₄₁₀N₆₄O₇₉S₆ requires 6081.1.

Example 18 Synthesis ofN^(εB29)-{4-[(15-carboxypentadecanoylamino)benzoyl]-desB30 human insulin

Step 1: 4-(15-tert-Butoxycarbonylpentadecanoylamino)-benzoic acid

Mono-tert-butyl hexadecandioate hexadecadioic acid (400 mg, 1.17 mmol)was dissolved in NMP (6 ml).1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (223 mg,1.17 mmol) and 1-hydroxy-7-azabenzotriazole (156 mg, 1.17 mmol) wasadded and the mixture was heated to 50 degrees celcius for 90 min.4-aminobenzoic acid (320 mg, 2.34 mmol) and DIEA (0.6 mL, 3.51 mmol) wasadded and the mixture was stirred under nitrogenflow overnight. Themixture is transferred is separated between saturated aqueous NaHCO₃ (50mL) and diethyl ether (100 mL). NaHSO₄ (50 mL, 10% in water) was addedand the organic phase isolated, dried (MgSO₄) and solvent removed invacuo. The crude product was recrystallized from ethanol to give4-(15-tert-butoxycarbonylpentadecanoylamino)-benzoic acid.

¹H-NMR (DMSO-d₆) δ: 10.18 (s, 1H), 7.87 (d, 2H), 7.69 (d, 2H), 2.33 (t,2H), 2.15 (t, 2H), 1.58 (t, 2H), 1.46 (t, 2H), 1.38 (s, 9H)_(m)1.31-1.20 (m, 20H).

Step 2: 4-(15-tert-Butoxycarbonyl-pentadecanoylamino)-benzoic acid2,5-dioxo-Pyrrolidin-1-yl ester

4-(15-tert-Butoxycarbonylpentadecanoylamino)-benzoic acid (29 mg, 0.063mmol) was converted to the succinimidyl ester using TSTU similar todescribed above.

HPLC-MS, m/z=559 (M+23, M+Na).

Step 3: N^(εB29)-{4-[(15-carboxypentadecanoylamino)benzoyl]-desB30 humaninsulin

DesB30 human insulin (355 mg, 0.062 mmol) was dissolved in DMSO (3.5mL), triethylamin (0.622 mmol, 0.087 mL) was added.4-(15-tert-Butoxycarbonyl-pentadecanoylamino)-benzoic acid2,5-dioxo-pyrrolidin-1-yl ester (28.3 mg, 0.051 mmol) was dissolved inDMSO (0.5 mL and added). The mixture was carefully stirred for 30minutes at room temperature. The mixture was frozen by cooling with anicebath and water (7 mL) was added and the mixture left standing at roomtemperature until the frozen mixture had dissolved. pH was adjusted topH=5.3 using 1 N HCl and precipitate isolated by centrifuge, washed oncewith water followed by centrifuge. Trifluoroacetic acid (15 mL) wasadded and the mixture was stirred for 30 minutes, poured into diethylether (50 mL) while maintaining cooling on an icebath. The crude productwas isolated by centrifuge and dissolved in 10 mM TRIS+15 mM (NH₄)₂SO₄in 20% EtOH, pH 7.3 and subjected to purification on an ÄKTA purifieremploying a reversed phase HPLC, Jupiter 5269, C4 250/20 mm, 15 μM, 300Å. The buffer consisted of A-buffer 10 mM TRIS+15 mM (NH₄)₂SO₄ in 20%EtOH, pH 7.3 and a B-buffer 80% EtOH. The product was eluted with agradient 15-60% B with 8 ml/min. Fractions containing product werecollected an pH adjusted to pH=5.2.N^(εB29)-{4-[(15-carboxypentadecanoylamino)benzoyl]-desB30 human insulinwas isolated by centrifuges and lyophilized.

LCMS: 6092.0, C₂₇₆H₄₀₉N₆₅O₇₉S₆ requires 6094.1.

Example 19 Synthesis ofN^(εB29)-{4-[(15-Carboxy-pentadecanoylamino)-methyl]-benzoyl}-desB30insulin

Step 1: 4-[(15-tert-Butoxycarbonylpentadecanoylamino)methyl]benzoic acid

Hexadecanedioic acid tert-butyl ester 2,5-dioxo-pyrrolidin-1-yl ester(370 mg, 0.842 mmol) was dissolved in NMP (8 mL), 4 aminomethyl benzoicacid (127.2 mg, 0.842 mmol) was added, the mixture was stirred at roomtemperature overnight, followed by heating at for 2 hours at 50 degreescelcius. After cooling to room temperature the mixture was poured intowater. Product isolated by filtration dried and used for the next stepwithout any further purification.

¹H-NMR (DMSO-d₆) (selected signals) δ: 7.88 (d, 2H), 7.34 (d, 2H), 4.31(d, 2H), 2.15 (m, 4H), 1.47 (m, 4H), 1.38 (s, 9H), 1.23 (br s, 2H), 1.38(s, 9H).

HPLC-MS: m/z=498 (M+Na)

Step 2:N^(εB29)-{4-[(15-Carboxy-pentadecanoylamino)-methyl]-benzoyl}-desB30insulin

The 4-[(15-tert-Butoxycarbonyl pentadecanoylamino)methyl]benzoic acidwas converted to converted to the succinimidyl ester and reacted withDesB30 human insulin similar to described above. The product waspurified on an ÄKTA purifier employing a reversed phase HPLC, Jupiter5269, C4 250/20 mm, 15 μM, 300 Å. The buffer consisted of A-buffer 10 mMTRIS+15 mM (NH₄)₂SO₄ in 20% EtOH, pH 7.3 and a B-buffer 80% EtOH. Theproduct was eluted with a gradient 15-60% B with 8 ml/min. Followed bypurification with RP-HPLC on a Waters Prep LC2000, on C18, 5 cm×20 cm,flow 20 ml/min using acetonitrile/water 36-42% gradient containing 0.1%TFA. Fractions containing product was collected and lyophilized. To thelyophilized material was added water (7.2 mL) and pH adjusted to 8.98with 1 N+0.1 N NaOH. The pH was adjusted back to 5.2-5.5 with 0.1 N HCl.The product precipitated, isolated by centrifuge and lyophilized to givethe title compound.

LCMS: 6108.0, C₂₇₇H₄₁₁N₆₅O₇₉S₆ 6108.1

Example 20 Hydrophobicity Albumin Affinity Self-Association andMixability of Long.Acting and Short-Acting Insulins Analysis ofSelv-Associating Properties of the Insulin Derivatives of the Invention

The ability of the insulin derivatives of the invention toself-associate into large, but soluble complexes is analysed using SEC(size exclusion chromatography):

Column: Superose ™ 6 PC 3.2/30, CV = 2.4 ml (Amerham Biosciences)Temperature: 37° C. SEC buffer: 140 mM NaCl, 10 mM TrisHCl, 0.01% NaN₃,pH 7.5 Injection volume: 20 μl Flow: 0.05 ml/min Runtime: 60 min andequillibration of additional 100 min

For this analysis the insulin derivatives of the invention are in asolution consisting of 0.6 mM derivative, 2.1 Zn²⁺/hexamer, 16 mMphenol, 7 mM phosphate pH 7.8. The retention time of the derivative isthen compared to the retention times of the following standardmolecules: Blue dextran (>5 MDa, K_(AV) 0.0), Thyroglobulin (669 kDa,K_(AV) 0.28), Ferritin (440 kDa, K_(AV) 0.39), Ovalbumin (44.5 kDa,K_(AV) 0.56), Ribonuclease (13.7 kDa, K_(AV) 0.69) and a secondreference of Albumin (66 kDa, K_(AV) 0.53), Co(III)insulin-hexamer (35kDa, K_(AV) 0.61), and monomeric insulin X2 (6 kDa, K_(AV) 0.73).

The following equation is used to determine the K_(av) for thederivative:

K _(av)=(t−t ₀)/(V _(t)/(f+t _(d) −t ₀))

Where t is the retention time for a given peak, t₀ is the retention timefor Blue dextran, V_(t) is the total column volume (here 2.4 ml), f isthe flow (here 0.04 ml/min), and t_(d) is the retention time for Bluedextran without the column in the system.

The K_(av) value indicates the degree of selv-association of aderivative, i.e. a large K_(av) similar to the K_(av) for theCo(III)insulin hexamer and X2 insulin monomer shows low or no propensityof the derivative to form large, selv-associated complexes, while verysmall K_(av) close to zero or even negative shows great propensity ofthe derivative for selv-association into large, soluble complexes.

Hydrophobicity Data on Insulin Derivatives According to the Invention.

The hydrophobicity (hydrophobic index) of the insulin derivatives of theinvention relative to human insulin, k′_(rel), was measured on aLiChrosorb RP18 (5 μm, 250×4 mm) HPLC column by isocratic elution at 40°C. using mixtures of A) 0.1 M sodium phosphate buffer, pH 7.3,containing 10% acetonitrile, and B) 50% acetonitrile in water aseluents. The elution was monitored by following the UV absorption of theeluate at 214 nm. Void time, t₀, was found by injecting 0.1 mM sodiumnitrate. Retention time for human insulin, t_(human), was adjusted to atleast 2t₀ by varying the ratio between the A and B solutions.k′_(rel)=(t_(derivative)−t₀)/(t_(human)−t₀). k′_(rel) found for a numberof insulin derivatives according to the invention are given in Table 1.

Human Serum Albumin Affinity Assay

Relative binding constant of 125I-TyrA14-analogue to human serum albuminimmobilised on Minileak particles and measured at 23° C. (detemir=1 insaline buffer).

Mixability of Long-Acting and Short-Acting Insulins as Analyzed bySize-Exclusion Chromatography of Insulin Mixtures

SEC: Mixability of Insulin Aspart (3 Zn/6 insulin, glycerol 1.6%, 16 mMphenol and 16 mM m-cresol, sodium chloride 10 mM, phosphate 7 mM, pH7.4) and prolonged acting insulin (2.1 or 6 Zn/6 insulin) 30:70, asmeasured by collecting fractions from SEC (as described above) andquantifying by HPLC the presence of prolonged-acting and fast-actinginsulins in the high molecular weight fraction (fraction 2, MW>HSA) andin the low molecular weight fraction (fraction 3, MW=HSA), respectively.

Four fractions are collected at size of 16 min after delay, of whichfraction 2 [16-32 min] (peak 1) contain associated form larger thanalbumin (32 min correspond to K_(AV) 0.46) and fraction 3 (peak 2)contain dihexameric, hexameric, dimeric and monomeric forms of insulin.

HPLC: Reverse phase chromatography on a Zorbax Eclipse XDB-C18 2.1*15 mm(1.8 μm) gradient eluted with buffer A: 0.2 M sodium sulphate, 0.04 Msodium phosphate, 10% acetonitrile, pH 7.2 and buffer B: 70%acetonitrile at 30° C., 19-34% B in 4.5 min. linear, sudden initialcondition at 5 min., run time of 7 min., flow of 0.5 ml/min., injectionvolume of 14 μL and UV detection at 276 nm using Insulin Aspartreference of 609 μM for both analogues.

Human Insulin serum receptor albumin Self- Hydrophobicity affinityaffinity association: relative to relative to relative to K_(av) humanhuman insulin (% area Compound insulin insulin detemir of peak) Example1 ++ ++ ++ +++ 1.366 37% 1.9 0.0  (87%) 0.74 (13%) Example 2 ++ ++ +++++ 1.933 35% 2.2 0.0  (94%) 0.75  (6%) Example 3 ++ ++ + +++ 1.095 30%0.37 0.08 (76%) 0.74 (24%) Example 4 +++ +++ + ++ 0.716 52%  0.069 0.15(77%) 0.74 (23%) Example 5 ++ ++ ++ +++ 1.75  30% 1.93 0.02 (93%) 0.76 (7%) Example 6 ++ ++ ++ +++ 1.617 33% 1.36 0.01 (90%) Example 7 ++ +++++ +++ 2.936 37% 2.62 0.01  (90%). Example 8 ++ ++ ++ n.a. 2.461 38%1.95 Example 9 +++ ++ + +++ 0.738 58% 0.06 0.05 (90%) 0.75 (10%) Example10 ++ ++ + ++ 1.803 43% 0.33 0.26 (48%) Example 11 +++ ++ n.a. n.a.0.435 43% Example 12 +++ +++ + ++ 0.989 49% 0.25 0.17 (44%) 0.7  (56%)Example 13 +++ +++ + + 0.552 59% 0.03 0.69 (100%)  Example 14 ++ ++ n.a.n.a. 1.61  48% Example 15 ++ ++ + ++ 1.09  47% 0.14 0.28 (42%) 0.72(58%) Example 16 ++ +++ ++ ++ 2.21  88% 1.9  0.3  (28%) 0.52 (72%)Example 17 + ++ +++ ++ 15.9   12% 2.97 0.12 (87%) 0.79 (13%) Example 18++ ++ +++ ++ 4.16  36% 5.83 0.33 (24%) 0.5  (76%) Example 19 ++ ++ +++++ 3.73  42% 5.27 0.33 (24%) 0.51 (76%) Table legend: Hydrophobicityrelative to human insulin: k'rel < 1: +++, 1-10: ++, >10: + (HI = 1)Insulin receptor affinity relative to human insulin: <5%: +, 5-50%:++, >50%: +++ Human serum albumin affinity relative to insulin detemir:<0.5: +, 0.5-2: ++, >2: +++ Self-association: K_(av) < 0.1: +++, K_(av)< 0.55: ++ and K_(av) ≧ 0.55: + K_(av) = 0.55 for human serum albumin,K_(av) = 0.63 for human insulin Co(III)hexamer, K_(av) = 0.72 for themonomeric insulin analogue X2. n.a. = not analyzed.

Example 21 Euglycaemic Glucose Clamp after s.c. Administration ofInsulin Preparations to Pigs

Female pigs, 60-90 kg, fasted for 18 h. During the experiments the pigsare free to move in their pens. An insulin dose is administered s.c.,depending of dose size often divided in two depots. Each pig is kepteuglycaemic at its individual fasting glucose levels for up to 24 h by avariable rate intravenous infusion of a 20% glucose solution. Theinfusion is given through a catheter inserted in the jugular vein.Depending on changes in plasma glucose concentrations observed duringfrequent plasma glucose monitoring, the necessary adjustments of theglucose infusion are made empirically. Blood samples are collected inEDTA glass tubes every 15-30 min, plasma separated for glucose andinsulin measurements. Glucose is determined within 1.5 min of bloodsampling with an YSI (Yellow Springs Instruments) glucose analyser(glucose oxidase method). Mean glucose infusion rate (GIR) profiles andmean plasma insulin profiles are made for each insulin preparation.(FIGS. 2-4 show mean±SEM).

Pharmacological Methods Assay (I) Insulin Receptor Binding of theInsulin Derivatives of the Invention

The affinity of the insulin analogues of the invention for the humaninsulin receptor was determined by a SPA assay (Scintillation ProximityAssay) microtiterplate antibody capture assay. SPA-PVT antibody-bindingbeads, anti-mouse reagent (Amersham Biosciences, Cat No. PRNQ0017) weremixed with 25 ml of binding buffer (100 mM HEPES pH 7.8; 100 mM sodiumchloride, 10 mM MgSO₄, 0.025% Tween-20). Reagent mix for a singlePackard Optiplate (Packard No. 6005190) is composed of 2.4 μl of a1:5000 diluted purified recombinant human insulin receptor-exon 11, anamount of a stock solution of A14 Tyr[¹²⁵I]-human insulin correspondingto 5000 cpm per 100 μl of reagent mix, 12 μl of a 1:1000 dilution of F12antibody, 3 ml of SPA-beads and binding buffer to a total of 12 ml. Atotal of 100 μl was then added and a dilution series is made fromappropriate samples. To the dilution series was then added 100 μl ofreagent mix and the samples were incubated for 16 hours while gentlyshaken. The phases were the then separated by centrifugation for 1 minand the plates counted in a Topcounter. The binding data were fittedusing the nonlinear regression algorithm in the GraphPad Prism 2.01(GraphPad Software, San Diego, Calif.).

Assay (II) Potency of the Insulin Derivatives of the Invention Relativeto Human Insulin

Sprague Dawley male rats weighing 238-383 g on the experimental day areused for the clamp experiment. The rats has free access to feed undercontrolled ambient conditions and fast overnight (from 3 pm) prior tothe clamp experiment.

Experimental Protocol

The rats are acclimatized in the animal facilities for at least 1 weekprior to the surgical procedure. Approximately 1 week prior to the clampexperiment Tygon catheters are inserted under halothane anaesthesia intothe jugular vein (for infusion) and the carotid artery (for bloodsampling) and exteriorised and fixed on the back of the neck. The ratsare given Streptocilin vet. (Boehringer Ingelheim; 0.15 ml/rat, i.m.)post-surgically and placed in an animal care unit (25° C.) during therecovery period. In order to obtain analgesia, Anorphin (0.06 mg/rat,s.c.) is administered during anaesthesia and Rimadyl (1.5 mg/kg, s.c.)is administered after full recovery from the anaesthesia (2-3 h) andagain once daily for 2 days.

The clamp technique employed is adapted from (1). At 7 am on theexperimental day overnight fasted (from 3 pm the previous day) rats areweighed and connected to the sampling syringes and infusion system(Harvard 22 Basic pumps, Harvard, and Perfectum Hypodermic glasssyringe, Aldrich) and then placed into individual clamp cages where theyrest for ca. 45 min before start of experiment. The rats are able tomove freely on their usual bedding during the entire experiment and hadfree access to drinking water. After a 30 min basal period during whichplasma glucose levels were measured at 10 min intervals, the insulinderivative to be tested and human insulin (one dose level per rat, n=6-7per dose level) were infused (i.v.) at a constant rate for 300 min.Plasma glucose levels are measured at 10 min intervals throughout andinfusion of 20% aqueous glucose is adjusted accordingly in order tomaintain euglyceamia. Samples of re-suspended erythrocytes were pooledfrom each rat and returned in about ½ ml volumes via the carotidcatheter.

On each experimental day, samples of the solutions of the individualinsulin derivatives to be tested and the human insulin solution aretaken before and at the end of the clamp experiments and theconcentrations of the peptides were confirmed by HPLC. Plasmaconcentrations of rat insulin and C-peptide as well as of the insulinderivative to be tested and human insulin are measured at relevant timepoints before and at the end of the studies. Rats are killed at the endof experiment using a pentobarbital overdose.

Test compounds and doses: Insulins to be tested are diluted from a stocksolution containing 97 μM of the insulin derivative in 5 mM phosphate pH7.7. The final concentration in the solution ready for use is 0.45 μM ofthe insulin derivative, 5 mM of phosphate, 100 mM of sodium chloride,0.007% of polysorbate 20. The pH was 7.7 and the i.v. infusion rate was15 and 20 pmol·min⁻¹·kg⁻¹.

A stock solution of human insulin that is used as reference compound wasformulated in a similar medium and infused i.v. at 6, 15 or 30pmol·min⁻¹·kg⁻¹.

Both stock solutions are stored at −20° C. and thawed overnight at 4° C.before use. The solutions are gently turned upside down several times 15min before they are transferred to the infusion syringes.

Assay (III)

Determination in pigs of T_(50%) of the insulin derivatives of theinvention

T_(50%) is the time when 50% of an injected amount of the A14 Tyr[¹²⁵I]labelled derivative of an insulin to be tested has disappeared from theinjection site as measured with an external γ-counter.

The principles of laboratory animal care are followed, Specificpathogen-free LYYD, non-diabetic female pigs, cross-breed of DanishLandrace, Yorkshire and Duroc, are used (Holmenlund, Haarloev, Denmark)for pharmacokinetic and pharmacodynamic studies. The pigs are conscious,4-5 months of age and weighing 70-95 kg. The animals fast overnight for18 h before the experiment.

Formulated preparations of insulin derivatives labelled in TyrA¹⁴ with¹²⁵I are injected sc. in pigs as previously described (Ribel, U.,Jørgensen, K, Brange, J, and Henriksen, U. The pig as a model forsubcutaneous insulin absorption in man. Serrano-Rios, M and Lefèbvre, P.J. 891-896.1985. Amsterdam; New York; Oxford, Elsevier SciencePublishers. 1985 (Conference Proceeding)).

At the beginning of the experiments a dose of 60 nmol of the insulinderivative according to the invention (test compound) and a dose of 60nmol of insulin detemir (both ¹²⁵I labelled in Tyr A14) are injected attwo separate sites in the neck of each pig.

The disappearance of the radioactive label from the site of sc.injection is monitored using a modification of the traditional externalgamma-counting method (Ribel, U. Subcutaneous absorption of insulinanalogues. Berger, M. and Gries, F. A. 70-77 (1993). Stuttgart; NewYork, Georg Thime Verlag (Conference Proceeding)). With this modifiedmethod it is possible to measure continuously the disappearance ofradioactivity from a subcutaneous depot for several days using cordlessportable device (Scancys Laboratorieteknik, Værløse, DK-3500, Denmark).The measurements are performed at 1-min intervals, and the countedvalues are corrected for background activity.

1. An insulin derivative having a formula

wherein Ins is a parent insulin moiety andQ₁-Q₂-[CH₂]_(n)-X₁-[CH₂]_(n)-Q₃-[CH₂]_(n)—X₂—[CH₂]_(n)-Q₄-[CH₂]_(n)-X₃—[CH₂]_(n)-Q₅-[CH₂]_(n)-Zis a substituent and where the Ins is attached to the substituent via anamide bond between the α-amino group of the N-terminal amino acidresidue of the B chain of Ins or an ε-amino group of a Lys residuepresent in the A or B chain of Ins and a CO group in Q₁ or Q₂ of thesubstituent; each n is independently 0, 1, 2, 3, 4, 5 or 6; Q₁ is: anamino acid amide of an amino acid with a carboxylic acid in the sidechain, or an amino acid with an uncharged side chain, which residueforms, with its carboxylic acid group, an amide group together with theα-amino group of the N-terminal amino acid residue of the B chain of Insor together with the ε-amino group of a Lys residue present in the A orB chain of Ins, or a chain composed of two, three or four α-amino acidamide or amino acid residues as specified above linked together viaamide bonds, which chain—via an amide bond—is linked to the α-aminogroup of the N-terminal amino acid residue of the B chain of Ins or tothe ε-amino group of a Lys residue present in the A or B chain of Ins,or a bond Q₂ is: —COCH(CONH₂)— —COCH₂N(CH₂CONH₂)——COCH₂N(CH₂CONH₂)COCH₂N(CH₂CONH₂) —COCH₂CH₂N(CH₂CH₂CONH₂)——COCH₂CH₂N(CH₂CH₂CONH₂)—COCH₂CH₂N(CH₂CH₂CONH₂)— —COCH₂N(CH₂CH₂CONH₂)——COCH₂CH₂N(CH₂CONH₂)— —COCH₂OCH₂CONH— —CO—((CR⁵R⁶)₁₋₆—NH—CO)₁₋₄—;—CO—((CR⁵R⁶)₁₋₆—CO—NH)₁₋₄—, where R⁵ independently can be H, —CH₃,—(CH₂)₁₋₆CH₃ or —CONH₂ and R⁶ independently can be H, —CH₃,—(CH₂)₁₋₆CH₃; or a bond provided that at least one of Q₁ or Q₂ is not abond, and that Q₂ is not —CO—(CH₂)₂ CO—NH— when n is 0 or 1, X₁ is abond and Q₃ is (CH₂CH₂O)₂—, (CH₂CH₂O)₃— or (CH₂CH₂OCH₂CH₂CH₂CH₂O)— andthat if an amine in Q₁ or Q₂ forms a bond with the rest of thesubstituent, the amine must be bound to the rest of the substituent viaa carbonyl group; Q₃, Q₄, and Q₅ independently of each other can be—(CH₂)_(m)— where m is an integer in the range of 6 to 32; a divalenthydrocarbon chain comprising 1, 2 or 3 —CH═CH— groups and a number of—CH₂— groups sufficient to give a total number of carbon atoms in thechain in the range of 4 to 32; —CO—((CR⁵R⁶)₁₋₆—NH—CO)—;—(CO—(CR⁵R⁶)₁₋₆—CO—NH)₁₋₄—, where R⁵ independently can be H, —CH₃,—(CH₂)₁₋₆CH₃ or —CONH₂ and R⁶ independently can be H, —CH₃,—(CH₂)₁₋₆CH₃; —CO—(CH₂)₀₋₃—Ar—(CH₂)₀₋₃— where Ar can be arylene orheteroarylene, which may be substituted with one or two groups selectedfrom the group consisting of —CH₃, —(CH)₁₋₆—CH₃, —CONR¹R² or —SO₂NR¹R²,where R¹ and R², independently of each other can be H, —CH₃ or—(CH)₁₋₆—CH₃; (CH₂CH₂O)_(y); (CH₂CH₂CH₂O)_(y)—; (CH₂CH₂CH₂CH₂O)_(y)—;(CH₂CH₂OCH₂CH₂CH₂CH₂O)_(y)— or (CH₂CH₂CH₂OCH₂CH₂CH₂CH₂O)_(y)—;—(CH₂OCH₂)_(y)— where y is 1-20; arylene or heteroarylene, which may besubstituted with one or two groups selected from the group consisting of—CH₃, —(CH)₁₋₆—CH₃, —CONR¹R² or —SO₂NR¹R², where R¹ and R²,independently of each other can be H, —CH₃ or —(CH)₁₋₆—CH₃; a chain ofthe formula—(CH₂)_(s)—Y₁—(Ar)_(v1)—Y₂—(CH₂)_(w)—Y₃—(Ar)_(v2)—Y₄—(CH₂)_(t)—Y₅—(Ar)_(v3)—Y₆—(CH₂)_(z)—wherein Ar is defined as above, Y₁-Y₆ independently of each other can beO, S, S═O, SO₂ or a bond; where s, w, t and z independently of eachother are zero or an integer from 1 to 10 so that the sum of s, w, t andz is in the range from 4 to 30, and v₁, v₂, and v₃ independently of eachother can be zero or 1 with the proviso that Y₁-Y₆ do not link to eachother and that the structure —O—(CH₂)₁—O— does not occur; or a bond;with the proviso that at least one of Q₃-Q₅ is not a bond; X₁, X₂ and X₃are independently of each other O; —C═O a bond; NCOR¹, where R¹ can beH, —CH₃ or —(CH)₁₋₆—CH₃; or

where R is hydrogen, C₁-3-alkyl, C₂₋₃-alkenyl or C₂₋₃-alkynyl; with theproviso that X₁, X₂ and X₃ cannot bind to Z and when X₁, X₂ and X₃ areO, then X₁, X₂ and X₃ do not bind directly to O in Q₃, Q₄, and Q₅ and Zis: —COOH; —CO-Asp; —CO-Glu; —CO-Gly; —CO-Sar; —CH(COOH)₂; —N(CH₂COOH)₂;—SO₃H —OSO₃H —OPO3H₂ —PO₃H₂ or -tetrazol-5-yl or —O—W₁, where W₁ isarylene or heteroarylene, which may be substituted with one or twogroups selected from the group consisting of tetrazo-5-lyl, —COOH,—SO₃H, —(CH₂)₁₋₆—SO₃H, —(CH₂)₁₋₆—O—PO₃H₂, —CONR³R⁴ or —SO₂NR³R⁴, whereR³ and R⁴, independently of each other can be H, —(CH₂)₁₋₆—SO₃H, or—(CH₂)₁₋₆—O—PO₃H₂; provided that when Z is —O—W₁ then Q₁ must be presentand any Zn²⁺ complex thereof.
 2. The insulin derivative according to aclaim 1, wherein Q₂ is selected from the group consisting of—COCH(CONH₂)— —COCH₂N(CH₂CONH₂)— —COCH₂N(CH₂CONH₂)COCH₂N(CH₂CONH₂)—COCH₂CH₂N(CH₂CH₂CONH₂)— —COCH₂CH₂N(CH₂CH₂CONH₂)—COCH₂CH₂N(CH₂CH₂CONH₂)——COCH₂N(CH₂CH₂CONH₂)— —COCH₂CH₂N(CH₂CONH₂)— —COCH₂OCH₂CONH—;—CO—((CR⁵R⁶)₁₋₆—NH—CO)₁₋₄—; or —CO—((CR⁵R⁶)₁₋₆—CO—NH)₁₋₄—, where R⁵independently can be H, —CH₃, —(CH₂)₁₋₆CH₃ or —CONH₂ and R⁶independently can be H, —CH₃, —(CH₂)₁₋₆CH₃
 3. The insulin derivativeaccording to claim 1, wherein Q₃ is —(CH₂)_(m)— where m is an integer inthe range of 6 to 32 or from 8 to 20 or m is 12, 13, 14, 15 or
 16. 4.The insulin derivative according to claim 1, wherein Q₁, Q₄, Q₅, X₁, X₂and X₃ is bonds and n is zero.
 5. The insulin derivative according toclaim 1, wherein one of Q₃, Q₄, or Q₅ is (CH₂CH₂O)_(y)—;(CH₂CH₂CH₂O)_(y)—; (CH₂CH₂CH₂CH₂O)_(y)—; (CH₂CH₂OCH₂CH₂CH₂CH₂O)_(y)— or(CH₂CH₂CH₂OCH₂CH₂CH₂CH₂O)_(y)—; —(CH₂OCH₂)— where y is 1-20.
 6. Theinsulin derivative according to claim 1, wherein Q₃ is—CO—((CR⁵R⁶)₁₋₆—NH—CO)—; —(CO—(CR⁵R⁶)₁₋₆—CO—NH)₁₋₄—, where R⁵independently can be H, —CH₃, —(CH₂)₁₋₆CH₃ or —CONH₂ and R⁶independently can be H, —CH₃, —(CH₂)₁₋₆CH₃; —CO—(CH₂)₃₋—Ar—(CH₂)₀₋₃—where Ar can be arylene or heteroarylene, which may be substituted withone or two groups selected from the group consisting of —CH₃,—(CH)₁₋₆—CH₃, —CONR¹R² or —SO₂NR¹R², where R¹ and R², independently ofeach other can be H, —CH₃ or —(CH)₁₋₆—CH₃; or a bond Q₄ is —(CH₂)_(m)—where m is an integer from 4 to 22; a divalent hydrocarbon chaincomprising 1, 2 or 3 —CH═CH— groups and a number of —CH₂— groupssufficient to give a total number of carbon atoms in the chain in therange of 4 to 22; arylene or heteroarylene, which may be substitutedwith one or two groups selected from the group consisting of —CH₃,—(CH)₁₋₆—CH₃, —CONR¹R² or —SO₂NR¹R², where R¹ and R², independently ofeach other can be H, —CH₃ or —(CH)₁₋₆—CH₃; or a chain of the formula—(CH₂)_(s)—Y₁—(Ar)_(v1)—Y₂—(CH₂)_(w)—Y₃—(Ar)_(v2)—Y₄—(CH₂)_(t)—Y₅—(Ar)_(v3)—Y₆—(CH₂)_(z)—wherein Ar is defined as above, Y₁-Y₆ independently of each other can beO, S, S═O, SO₂ or a bond; where s, w, t and z independently of eachother are zero or an integer from 1 to 10 so that the sum of s, w, t andz is in the range from 4 to 30, and v₁, v₂, and v₃ independently of eachother can be zero or 1 with the proviso that Y₁-Y₆ do not link to eachother and that the structure —O—(CH₂)₁—O— does not occur; X₁ is O; —C═ONCOR¹, where R¹ can be H, —CH₃ or —(CH)₁₋₆—CH₃; or

where R is hydrogen, C₁₋₃-alkyl, C₂₋₃-alkenyl or C₂₋₃-alkynyl; with theproviso that when X₁ is O, then X₁ does not bind directly to O in Q₄;X₂, X₃ and Q₅ are bonds; All values of n are zero; and Z is: —COOH;—CO-Asp; —CO-Glu; —CO-Gly; —CO-Sar; —CH(COOH)₂; —N(CH₂COOH)₂; —SO₃H—OSO₃H —OPO3H₂ —PO₃H₂ or -tetrazol-5-yl or —O—W₁, where W₁ is arylene orheteroarylene, which may be substituted with one or two groups selectedfrom the group consisting of tetrazo-5-lyl, —COOH, —SO₃H,—(CH₂)₁₋₆—SO₃H, —(CH₂)₁₋₆—O—PO₃H₂, —CONR³R⁴ or —SO₂NR³R⁴, where R³ andR⁴, independently of each other can be H, —(CH₂)₁₋₆—SO₃H, or—(CH₂)₁₋₆—O—PO₃H₂; and any Zn²⁺ complex thereof.
 7. The insulinderivative according to claim 1, wherein Z is —COOH.
 8. The insulinderivative according to claim 1, wherein the parent insulin is aninsulin analogue.
 9. The insulin derivative according to claim 8,wherein the parent insulin is selected from the group consisting of:desB30 human insulin, GlyA21 human insulin, GlyA21desB30 human insulin,GlyA21ArgB31ArgB32 human insulin, LysB3GluB29 human insulin,LysB28ProB29 human insulin and ThrB29LysB30 human insulin.
 10. Apharmaceutical composition for the treatment of diabetes in a patient inneed of such treatment, comprising a therapeutically effective amount ofan insulin derivative according to claim 1 together with apharmaceutically acceptable carrier.
 11. A method for producing apharmaceutical composition according to claim 10, wherein up to about 10zinc atoms per 6 molecules of insulin derivative are added to thepharmaceutical composition.
 12. A method of treating diabetes in apatient in need of such a treatment, comprising administering to thepatient a therapeutically effective amount of an insulin derivativeaccording to claim
 1. 13. The method according to claim 12 for pulmonarytreatment of diabetes.
 14. A mixture of an insulin derivative accordingto claim 1 and a rapid acting insulin analogue selected from the groupconsisting of: AspB28 human insulin, LysB28ProB29 human insulin andLysB3GluB29 human insulin.
 15. An insulin derivative, wherein theinsulin derivative is selected from the group consisting of:N^(εB29)-ω-carboxy-pentadecanoyl-γ-L-glutamylamide desB30 human insulin,N^(εB29)-ω-carboxy-pentadecanoyl-γ-amino-butanoyl desB30 human insulin,N^(εB29)-ω-carboxy-tetradecanoyl-γ-L-glutamylamide desB30 human insulin,N^(εB29)-ω-carboxy-tridecanoyl-γ-L-glutamylamide desB30 human insulin,N^(εB29)-ω-carboxy-pentadecanoyl-β-alanyl desB30 human insulin,N^(εB29)-ω-carboxy-pentadecanoyl-γ-L-aspartylamide desB30 human insulin,N^(εB29)-ω-carboxy-pentadecanoyl-ε-aminohexanoyl desB30 human insulin,N^(εB29)-ω-carboxy-pentadecanoyl-δ-aminopentanoyl desB30 human insulin,N^(εB29)-10-(4-carboxyphenoxy)-decanoyl-γ-L-glutamylamide desB30 humaninsulin, N^(εB29)-4-[11-(4-Carboxyphenyl)undecanoylamino]butyryl desB30human insulin,N^(εB29)-(3-(3-{4-[3-(7-carboxyheptanoylamino)propoxy]butoxy}propylcarbamoyl)-propionyl-γ-glutamylamide)desB30 human Insulin, N^(εB29)-ω-carboxy-tridecanoyl-γ-amino-butanoyldesB30 human insulin, N^(εB29)-ω-carboxy-undecanoyl-γ-amino-butanoyldesB30 human insulin, N^(εB29)-ω-carboxy-tetradecanoyl-γ-amino-butanoyldesB30 human insulin,N^(εB29)-{4-[10-(4-Carboxy-phenoxy)-decanoylamino]-butyryl} desB30insulin, N^(εB29)-{4-[(14-Carboxy-tetradecanoylamino)-methyl]-benzoyl}desB30 insulin, N^(εB29)-[16-(4-Carboxy-phenoxy)-hexadecanoyl] desB30insulin, N^(εB29)-4-[(15-carboxypentadecanoylamino)benzoyl]-desB30 humaninsulin andN^(εB29)-{4-[(15-Carboxy-pentadecanoylamino)-methyl]-benzoyl}-desB30insulin.